The biochemistry of cancer and nucleic acid metabolism … first ye… · The Biochemistry of...

Prof. Dr. Hedef Dhafir El-Yassin 2012

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The Biochemistry of Cancer and

Nucleic acids Metabolism 1. Lecture 1: DNA structure and replication 2. Lecture 2: DNA mutation and repair mechanisms 3. Lecture 3: RNA structure, transcription, post-transcriptional processing and drugs that inhibit these

processes. 4. Lecture 4: Protein synthesis and translation in prokaryotic and eukaryotic cells and drugs that

inhibit this process 5. Lecture 5: Post translational processes and protein folding 6. Lecture 6: Protein targeting and degradation 7. Lecture 7: Biochemistry of cancer and tumor markers 8. Lecture 8: The biochemistry of nucleic acids metabolism 9. Lecture 9: Clinical cases and biochemical interpretations (1) 10. Lecture 10: Clinical cases and biochemical interpretations (2)

Molecular cell biology is studying the molecular basis of biological activity. It is a multitalented, broad subject that can be studied from three different aspects: biology, biochemistry and pathology….and may be more depending on how to approach the subject. Aim and objective of the above ten lectures is to understand:

1. The constitution and general properties of the biochemistry of nucleic acids (DNA and (RNA)

2. The importance of regulation of DNA replication, mutation and repair mechanism to the biochemistry of cell cycle and how it impacts on understanding of human cancer.

3. How DNA repair complexes are assembled and to show how DNA damage response is triggered by the short telomeres of human cells undergoing replicative senescence.

4. RNA transcription and regulation and how it is involved in developing therapy for cancer treatment.

5. The control of gene expression and Molecular mechanism of protein synthesis,

6. Protein targeting and folding. Diseases generated from protein misfolding

7. The biochemistry of cancer

8. Tumor markers

References: 1. "Biochemistry" by Lubert Stryer (textbook) 2. "Textbook of Biochemistry with Clinical Correlations" by T.M.Devlin

(additional reading) 3. "Lippincott's Illustrated Reviews in Biochemistry" by P.C.Champe, R.A.Harvey and D.R.Ferrier

(additional reading) 4. "Harper's Biochemistry" by R.K.Murray, D.K.Granner, P.A. Mayes and V.W.Rodwell.

(additional reading) 5. "Clinical Laboratory Science Review" By Robert R. Harr (additional reading)


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Lecture 1: DNA structure and replication

GENE EXPRESSION Gene expression also called protein expression or often simply expression: is the process by which a gene's DNA sequence is converted into the structures and functions of a cell. Gene expression is a multi-step process that begins with transcription of DNA, which genes are made of, into messenger RNA. It is then followed by post transcriptional modification and translation into a gene product, followed by folding, post-translational modification and targeting.

The amount of protein that a cell expresses depends on: 1. the tissue, 2. the developmental stage of the organism 3. and the metabolic or physiologic state of the cell.

Structure of DNA 1. Primary structure of DNA

Although sometimes called "the molecule of Heredity", DNA are not single molecules. Rather, they are pairs of molecules, double helix . Each molecule is a strand of DNA: a chemically linked chain of nucleotides each of which consists of a sugar a phosphate and one of four kinds of aromatic hydrocarbon "nitrogen bases". Because DNA strands are composed of these nucleotide subunits, they are polymers. The diversity of the bases means that there are four kinds of nucleotides, which are commonly referred to by the identity of their bases. These are adenine (A), thymine (T), cytosine (C), and guanine (G).


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In all living cells, DNA serves to store genetic information. Specific segments of DNA (“genes”) are transcribed as needed into RNAs, which either carry out:

• structural • or catalytic tasks themselves • or provide the basis for synthesizing proteins.

In the latter case, the DNA codes for the primary structure of proteins. The “language” used in this process has four letters (A, G, C, and T). All of the words (“codons”) contain three letters (“triplets”), and each triplet stands for one of the 20 proteinogenic amino acids. The two strands of DNA are not functionally equivalent:

1. The template strand (the (–) strand or “codogenic strand,” shown in light gray in figure below) is the one that is read during the synthesis of RNA (transcription). Its sequence is complementary to the RNAformed.

2. The sense strand (the (+) strand or “coding strand,” shown in color in figure below has the same sequence as the RNA, except that T is exchanged for U.

Gene sequences are expressed by reading the sequence of the sense strand in the 5'-to-3' direction. Using the genetic code in this case the protein sequence(3 in the figure below) is obtained directly in the reading direction usual for proteins—i. e., from the N terminus to the C terminus.


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2. Secondary structure of DNA ( DNA: conformation) Investigations of synthetic DNA molecules have shown that DNA can adopt several different conformations. All of the DNA segments shown consist of 21 base pairs (bp) and have the same sequence. By far the most common form is B-DNA (2 in the figure below). This consists of two antiparallel poly-deoxynucleotide strands intertwined with one another to form a right-handed double helix. The “backbone” of these strands is formed by deoxyribose and phosphate residues linked by phosphoric acid diester bonds. In the B conformation, the aromatic rings of the nucleo-bases are stacked at a distance of 0.34 nm almost at right angles to the axis of the helix. Each base is rotated relative to the preceding one by an angle of 35°. A complete turn of the double helix (360°) therefore contains around 10 base pairs (abbreviation: bp), i. e., the pitch of the helix is 3.4 nm. Between the backbones of the two individual strands there are two grooves with different widths:

1. The major groove is visible at the top and bottom, 2. while the narrower minor groove is seen in the middle.

DNA-binding proteins and transcription factors usually enter into interactions in the area of the major groove, with its more easily accessible bases. In certain conditions, DNA can adopt the A conformation (1 in the figure below). In this arrangement, the double helix is still right-handed, but the bases are no longer arranged at right angles to the axis of the helix, as in the B form. As can be seen, the A conformation is more compact than the other two conformations. The minor groove almost completely disappears, and the major groove is narrower than in the B form. A-DNA arises when B-DNA is dehydrated. It probably does not occur in the cell. In the Z-conformation (3 in the figure below), which can occur within GC-rich regions of B-DNA, the organization of the nucleotides is completely different. In this case, the helix is left-handed, and the backbone adopts a characteristic zig-zag conformation (hence “Z-DNA”). The Z double helix has a smaller pitch than B-DNA.


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DNA segments in the Z conformation are methylated and probably have physiological significance, but details are not yet known.

3. Tertiary structure of DNA

The DNA of a single human cell, if stretched to its full length is 1.74 meters. To get DNA into a cell's nucleus it must be packaged into a more tightly compacted form. The structural flexibility of DNA allows it to adopt more compacted structures than simple linear B-form DNA.


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Expression and transmission of genetic information The genetic information of all cells is stored in the base sequence of their DNA (RNA only occurs as a genetic material in viruses. Functional sections of DNA that code for inheritable structures or functions are referred to as genes. Most genes code for proteins—i. e., they contain the information for the sequence of amino acid residues of a protein (its sequence).


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DNA REPLICATION DNA replication or DNA synthesis is the process of copying a double-stranded DNA strand, prior to cell division. The two resulting double strands are identical (if the replication went well), and each of them consists of one original and one newly synthesized strand. This is called semi conservative replication.

The process of replication consists of three steps, initiation, replication and termination.

1. Prokaryotic replication Basic Requirement for DNA Synthesis

1. Substrates: the four deoxy nucleosides triphosphates are needed as substrates for DNA synthesis. Cleavage of the high-energy phosphate bond between the α and β phosphates provides the energy for the addition of the nucleotide.

2. Template: DNA replication cannot occur without a template. A template is required to direct the addition of the appropriate complementary deoxynucleotide to the newly synthesized DNA strand.

3. Primer: DNA synthesis cannot start without a primer, which prepares the template strand for the addition of nucleotides.

4. Enzyme: the DNA synthesis that occurs during the process of replication is catalyzed by enzymes called DNA-dependent DNA polymerases. Commonly called DNA polymerases.


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Multiple DNA Polymerase with Multiple Enzymatic Activities.

DNA polymerase

A DNA polymerase is an enzyme that assists in DNA replication. Such enzymes catalyze the polymerization of deoxyribonucleotides alongside a DNA strand, which they "read" and use as a template. The newly polymerized molecule is complimentary to the template strand and identical to the template's partner strand.

All DNA polymerases synthesize DNA in the 5' to 3' direction. But no known DNA polymerase is able to begin a new chain. They can only add a nucleotide onto a preexisting 3'- OH group. For this reason DNA polymerase needs a primer at which it can add the first nucleotide.

DNA polymerase I: is an enzyme that aids in DNA replication. It was discovered in the mid 1950's, and was the first such enzyme discovered (hence the name). It is often referred to as Pol I, for short. DNA polymerase I removes the RNA primer from the lagging strand and fills in the necessary nucleotides. Ligase then joins the various fragments together into a continuous strand of DNA.

DNA polymerase II: is a minor DNA polymerase in E. coli, may be involved on some DNA repair processes. It is often referred to as Pol II, for short.

DNA polymerase III holoenzyme: Pol III is a holoenzyme that aids in DNA replication. As a replicative enzymatic mechanism of DNA, the Polymerase replicates with high fidelity.


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Origin of Replication

The origin of replication (also called replication origin or oriC) is a unique DNA sequence at which DNA replication is initiated and proceeds bidirectionally or unidirectionally.

1. OriC: The origin of replication oriC is a 250 bp sequence rich in adenine-thymine base pairs, which are more easily separated than cytosine-guanine base pairs.

2. DnaA: dnaA is an initiation factor which hydrolyzes ATP and promotes the unwinding or melting of DNA at oriC, during DNA replication. The oriC/dnaA complex formation does not require ATP until it is open.

After initiation, dnaA binds dnaB and dnaC.

3. Replication fork: The replication fork is a structure which forms when DNA is ready to replicate itself. It is created by topoisomerase, which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. DNA polymerase then goes to work on creating new partners for the two strands by adding nucleotides.

Basic Molecular Events at Replication Forks: 1. Leading strand synthesis: is the continuous synthesis of one of the daughter strands in

a 5' to 3' direction. Pol III catalyzes leading strand synthesis.


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2. Lagging strand synthesis:

a. Okazaki fragments: One of the newly synthesized daughter strands is made discontinuously. The resulting short fragments are called Okazaki fragments. These fragments are latter joined by DNA ligase to make a continuous piece of DNA. This is called lagging strand synthesis. Discontinuous synthesis of lagging strands occurs because DNA synthesis always occurs in a 5' to 3' direction. Pol III catalyzes lagging strand synthesis

b. Direction of new synthesis: As the replication fork moves forward, leading strand synthesis follows. A gap forms on opposite strand because it is in the wrong orientation to direct continuous synthesis of a new strand. After a lag period, the gap that forms is filled in by 5' to 3' synthesis. This means that new DNA synthesis on the lagging strands is actually moving away from the replication fork.

c. Priming of Okazaki fragment synthesis. i. Enzyme: an enzyme called primase is the catalytic portion of a primosome

that makes the RNA primer needed to initiate synthesis of Okazaki fragment. It also makes the primer that initiates leading strand synthesis at the origin.

ii. Primers provide a 3'-hydroxyl group that is needed to initiate DNA synthesis. The primers made by primase are small pieces of RNA (4-12 nucleotides) complementary to the template strand.

d. The role of pol I in replication: On completion of lagging strand synthesis by pol III, the RNA primer is then removed by pol I and replaced with DNA. Synthesis of each new Okazaki fragments takes place until it reach's the RNA primer of the preceding Okazaki fragment and the RNA primer. DNA pol I uses its nick-translation properties to hydrolyze the RNA (5' to 3' exonuclease activity) and replace it with DNA.

e. Joining of Okazaki fragments: After pol I has removed the RNA primer and replaced it with DNA, an enzyme called DNA ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is removed and filled in. The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.:


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Other Factors Needed for Propagation of Replication Forks 1. Topoisomerase is responsible for initiation of the unwinding of the DNA. 2. Helicases: are enzymes that catalyze the unwinding of the DNA helix. A helicase derives

energy from cleavage of high energy phosphate bonds of nucleoside triphosphates, usually ATP, to unwind the DNA helix. Hilcase activity provides single strand templates for replication:

3. Gyrase. : Positive supercoils would build up in advance of a moving replication fork without the action of gyrase, which is a topomerase.

4. single-strand binding protein (SSBP): a. Function: SSBP enhances the activity of helicase and binds to a single-strand

template DNA until it can serve as a template. It may also serve to protect single strand DNA from degradation by nucleases, and it may block formation of intrastrand duplexes of hairpins that can slow replication.

b. Release: SSBP is displaced from single strand DNA when the DNA undergoes replication.

5. Primosome a. Definition: the primosome is a complex of proteins that comprises primase, a hexamer

of the helicase dnaB protein, dnaC protein and several other proteins. b. Function: the primosome complex primes DNA synthesis at the origin. Driven by ATP

hydrolysis, the primosome moves with the replication fork, making RNA primes for Okazaki fragment synthesis.


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The Replisome: It is believed that all the replication enzymes and factors are part of a large macromolecular complex called replisome. It has been suggested that the replisome may be attached to the membrane and that instead of the replisome moving along the DNA during replication, DNA passed through the stationary replisome. Replosome model of replication Termination of Replication: Replication sequences (e.g. ter) direct termination for replication. A specific protein (the termination utilization substance (TUS) protein) binds to these sequences and prevents the helicase dnaB protein from further unwinding DNA. This facilitates the termination of replication.

2. Eukaryotic Replications Eukaryotes are organisms with complex cells, in which the genetic material is organized into membrane-bound nuclei They may utilize slightly different mechanisms of replication. However most of these mechanisms are very similar to those in prokaryotic replication. Replicons are basic units of replication.

1. Function: A replicon encompasses the entire DNA replicated from the growing replication forks that share a single origin.

2. Size: Replicons may vary in size from 50-120 μm. There are estimated to be 10,000-100,000 replicon per cell in mammals. The large number of replicons is needed to replicate the large mammalian genomes in a reasonable period of time. It takes approximately 8 hours to replicate the human genome.

3. Replication rate: a. Prokaryotes. An E. coli replication fork progresses at approximately 1000 base

pairs per second. b. Eukaryotes. The eukaryotic replication rate is about 10 times slower than the

prokaryotic replication rate. Each replicon complete synthesis in approximately an hour. Therefore during the total period of eukaryotic replication not every replicon is active. The slow rate of eukaryotic replication is likely due to interferences of nucleusomes and chromosomal proteins.


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Multiple Eukaryotic DNA Polymerases

1. DNA polymerase alpha : This enzyme is composed of 4 subunits, one of which (167 kDa) carries the polymerase activity. It is responsible for synthesis of the primer on the lagging strand because it is responsible for the initiation of Okazaki fragments. The primer consists of both RNA and a short stretch (20 nt) of DNA.

2. DNA polymerase delta : This enzyme contains at least 4 and maybe as many as a dozen subunits. It has a proofreading activity. When associated with proliferating cell nuclear antigen (PCNA), it has a very high processivity.

3. DNA polymerase beta & DNA polymerase epsilon: Both enzymes are involved in DNA repair.

4. DNA polymerase gamma: This enzyme is located in the mitochondrion where it is responsible for replication of mtDNA.

Telomere

A telomere is a region of highly repetitive DNA at the end of a chromosome, which functions as an aglet. If it were not for telomeres, this would quickly result in the loss of useful genetic information.

In prokaryotes, chromosomes are circular and thus do not have ends to suffer premature replication termination at. Only eukaryotes possess or require telomeres.


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Telomeres are extended by telomerases, Telomerases are very interesting DNA polymerases in that they carry an RNA template for the telomere sequence within them.

• Structure of telomeres: In humans, the telomere sequence is a repeating string of TTAGGG, between 3 and 20 kilobases in length. There are additional 100-300 kilobases of telomere-associated repeats between the telomere and the rest of the chromosome. Telomere sequences vary from species to species, but are generally GC-rich.

• The mechanism of telomere replication: Telomerase provide an RNA template complementary to the telomeric repeat, and the free 3' end of the telomere is the primer for new DNA synthesis. After elongation of the telomere by telomerase, normal lagging strand synthesis presumably makes a complementary copy of all but the 3' most terminal sequences.

In most multicellular eukaryotes, telomerase is only active in germ cells. There are theories that the steady shortening of telomeres with each replication in somatic (body) cells may have a role in senescence and in the prevention of cancer.

Clinical relevance of telomeres: If telomeres become too short, they will uncap. The cell will detect this as DNA damage and will enter cellular senescence (growth arrest). Uncapped telomeres also result in chromosomal fusions. Since this damage cannot be repaired in normal somatic cells, the cell may even go into apoptosis. Many aging-related diseases are linked to shortened telomeres. Organs deteriorate as more and more of their cells die off or enter cellular senescence.

Cancer

When normal cells are damaged or old they undergo apoptosis; cancer cells, however, avoid apoptosis. All cancers begin in cells and are caused by mutations. Normally, cells grow and divide to form new cells only when the body needs them. When cells grow old and die, new cells take their place. Mutations can sometimes disrupt this orderly process. New cells form when the body does not need them, and old cells do not die when they should.


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Lecture 2: DNA mutation and repair mechanisms

DNA MUTATIONS AND THEIR REPAIR

MUTATIONS: are any permanent changes in the genetic material (usually DNA or RNA) of a cell.

Mutations can be caused by copying errors in the genetic material during cell division and by exposure to radiation, chemicals, or viruses, or can occur deliberately under cellular control during the processes such as meiosis or hypermutation.

In multicellular organisms, mutations can be subdivided into germline mutations, which can be passed on to progeny and somatic mutations, which (when accidental) often lead to the malfunction or death of a cell and can cause cancer.

Mutations are considered the driving force of evolution, where less favorable (or deleterious) mutations are removed from the gene pool by natural selection, while more favorable (or beneficial) ones tend to accumulate.

Mutagenesis is the process by which mutations arise. Both words originate from the Latin mutare, to change.

Types of mutations

• Point mutations are usually caused by chemicals or malfunction of DNA replication and exchange a single nucleotide for another. Most common is the transition that exchanges a purine for a purine or a pyrimidine for a pyrimidine (A ↔ G, C ↔ T). A transition can be caused by nitrous acid, base mispairing, or mutagenic base analogs such as 5-bromo-2-deoxyuridine (BrdU). Less common is a transversion, which exchanges a purine for a pyrimidine or a pyrimidine for a purine (C/T ↔ A/G). A point mutation can be reversed by another point mutation, in which the nucleotide is changed back to its original state (true reversion) or by second-site reversion (a complementary mutation elsewhere that results in regained gene functionality). Point mutations are called silent, missense or nonsense mutations, depending on whether the erroneous codon codes for the same amino acid (silent), a different amino acid (missense) or a stop, which can truncate the protein (nonsense).

• Insertions add one or more extra nucleotides into the DNA. They are usually caused by transposable elements, or errors during replication of repeating elements (e.g. AT repeats). Most insertions in a gene can cause a shift in the reading frame (frameshift) or alter splicing of the mRNA, both of which can significantly alter the gene product. Insertions can be reverted by excision of the transposable element.

• Deletions remove one or more nucleotides from the DNA. Like insertions, these mutations can alter the reading frame of the gene. They are irreversible.


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Original DNA molecule ACGAGTGTGCGATCACCT Transcription

Insertion of extra T unit

mRNA UGCUCACACGCUAGUGGA

Translation

Peptide Cys Ser His Ala Ser Gly Extra unit

Mutant DNA ACGATGTGTGCGATCACCT

Transcription

Mutant mRNA UGCUACACACGCUAGUGGA

Translation

Mutant peptide Cys Tyr Thr Arg The mutant peptide not only has the incorrect order of amino acids but also shorter.

Causes of mutation Two classes of mutations are spontaneous mutations (naturally occurring) and induced mutations caused by mutagens.

Spontaneous mutations on the molecular level include:

a. Errors in replication. If a base that is noncomplementory to the template base added during replication, then a mispairing or mismatch occurs. This leads to a mutation during the next round of replication if the error is not repaired.

b. Errors that occur during recombination events. (Recombinant DNA: molecules of DNA formed by inserting portions of DNA from one organism into DNA of another.

c. Tautomerism a. Keto ↔ Enol b. Amino ↔ Imino


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An example of a spontaneous transition results from the tautomerization of adenine to generate a form, which can base pair with cytosine. An amino group (-NH2) tautomerizes to an imino group (=NH) (other tautomers can form from a keto group (-C=O) changing to an enol group (-C-OH)):

d. Substitutions - one base is replaced by another. If this mutation occurs within the coding sequence of a gene, it may lead to the use of a different amino acid or generate a stop codon. Substitutions fall into two categories:

o Transitions - one purine is replaced by another (A -> G or G -> A), or one pyrimidine is replaced by another (C -> T or T -> C)

o Transversions - a purine is replaced by a pyrimidine (A -> C or T; G -> C or T), or a pyrimidine is replaced by a purine (C -> A or G; T -> A or G)

e. Frameshift mutation (insertion or deletion on one strand), usually through a polymerase error when copying repeated sequences

a. Deletions - one or more bases is removed. Unless this mutation results in the loss of a multiple of three bases, a frame-shift will occur in coding sequences, drastically altering every codon downstream of the mutation, and therefore the final amino acid composition of the protein.

b. Insertions - one or more bases is added. The effects are the same as deletions, resulting in frame-shift mutations.

f. Oxidative damage caused by oxygen radicals g. Spontaneous changes:

a. Deamination of cytosine (C) to form uracil (U). b. Spontaneous depurination. Purines are less stable under normal cellular

conditions than pyrimidines. The glycosidic bond that links purines to the


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sugar-phosphate backbone of DNA often is broken. If these purines are not replaced before a round of replication, any base may be added to complement the missing base during replication.

Induced mutations on the molecular level include:

1. Chemical mutations 1. Nonalkylating agents. For example:

i. Formaldehyde (HCHO) reacts with amine groups and cross-links DNA, RNA and proteins.

ii. Hydroxylamine (NH2OH) specifically reacts with cytosine to form derivatives that pair with adenines instead of guanines. This change lead to a transversion (in which a purine is replaced by a pyrimidine or a pyrimidine is replaced by a purine.

iii. Nitrous acid (HNO2) oxidatively deaminates cytosine, adenines, and guanines to form uracil, hypoxanthines, and xanthines respectively. These changes results in transitions (in which a purine is replaced by another purine or one pyrimidine is replaced by another pyrimidine.

b. Alkylating agents: these act as strong electrolytes, which become linked to many cellular nucleophiles in particular the sevenths nitrogen of the guanine in the DNA. This linkage causes breakage of DNA.

2. Irradiation a. Ultraviolet (UV) light (200-400 nm) induces dimerization of adjacent

pyrimidines, particularly adjacent thymines. This direct mutation of DNA distorts the DNA structure, inhibits transcription, and disrupts replication until it is repaired.

b. Ionizing radiation, such as Roentgen rays (x rays) and gamma rays (γ-rays) can cause extensive damage to DNA including opening purine rings, which lead to depurination, and breaking phosphodiester bonds.

DNA has so-called hotspots, where mutations occur up to 100 times more frequently than the normal mutation rate. A hotspot can be at an unusual base, e.g., 5-methylcytosine.

Mutation rates also vary across species. Evolutionary Biologists have theorized that higher mutation rates are beneficial in some situations, because they allow organisms to evolve and therefore adapt faster to their environments.

Some mutagens chemically modify the DNA bases. Nitrous oxide deaminates adenine to hypoxanthine which base-pairs with cytosine, it also deaminates cytosine to uracil which base-pairs to adenine. Hydroxylamine converts cytosine to a form which base-pairs with adenine, causing specific transitions from C-G to T-A. Flat, aromatic compounds such as the acridines intercalate into the DNA helix, inserting themselves between adjacent bases. This can lead to the insertion or deletion of one or more base pairs. Ethidium bromide, a reagent used in molecular biology, intercalates into DNA. This compound fluoresces under UV light, allowing the visualization of DNA in an agarose gel.


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DNA repair Some mutations in DNA can be repaired because the genetic information is stored on both strands of DNA. The unaffected strand can be used as a template to fix the damaged strand. Chemicals, ionizing radiation and ultraviolet light can cause breakage of the phosphodiester bonds in the DNA backbone, and the bases themselves can be altered, lost, or covalently cross-linked.

UV light can cause adjacent pyrimidine bases to become covalently joined, forming a pyrimidine dimer.

This lesion is removed by an excinuclease, an enzyme which excises a 12 bp fragment surrounding the dimer. DNA polymerase I fills in the gap and DNA ligase seals the break:


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An alternative mechanism to repair pyrimidine dimers uses the enzyme DNA photolyase, which uses light energy to cleave the dimer back into the original bases.

An important aspect in the repair of DNA, especially in base mismatches, is the ability to distinguish between strands. Parental DNA can be distinguished from the newly synthesized strand by methylation of adenine residues. Specific methylases react with adenine in GATC sequences. This enzyme takes time to operate, so in newly synthesized DNA the daughter strand won't be methlyated and the repair mechanisms can identify the parental strand and use it as a template to correct the unmethylated strand.

Defects in the repair mechanism of DNA can lead to cancer.

Xeroderma pigmentosum can result from a deficiency in the excinuclease which removes pyrimidine dimers. Individuals with this disease frequently die from metastases of malignant skin tumors before the age of 30.

Defective mismatch repair can result in hereditary nonpolyposis colorectal cancer . Mutations build up in the genome over time until eventually a gene controlling cell proliferation is altered, resulting in a cancerous tumor.


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Potential carcinogens can be identified by the use of a test on bacteria because many carcinogens and mutagens exert their effects on DNA. Salmonella which have a mutation in their histidine biosynthetic pathway are plated onto a medium lacking in histidine. Addition of mutagenic agents to this medium results in the development of revertants, strains which are capable of growth on this medium. By changing the specific mutation in the orginal strain, in is possible to distinguish agents which cause base-pair substitutions from agents which cause frame-shift mutations. The addition of mammalian liver homogenate expands the sensitivity to mutagenic agents which result from the conversion of a precursor form. The bacterial cells lack the enzyme systems which produce some of these compounds during their degradation in the liver.

The DNA repair process must be constantly operating, to correct rapidly any damage in the DNA structure.

As cells age, however, the rate of DNA repair can no longer keep up with ongoing DNA damage. The cell then suffers one of three possible fates:

1. an irreversible state of dormancy, known as senescence 2. cell suicide, also known as apoptosis or programmed cell death 3. cancer

Most cells in the body become senescent. Then, after irreparable DNA damage, apoptosis occurs. In this case, apoptosis functions as a "last resort" mechanism to prevent a cell from becoming cancerous and endangering the organism.

When cells become senescent, alterations in their gene regulation cause them to function less efficiently, which inevitably causes disease. The DNA repair ability of a cell is vital to its normal functioning and to the health and longevity of the organism.

Nuclear versus mitochondrial DNA damage In human, and eukaryotic cells in general, DNA is found in two cellular locations - inside the nucleus and inside the mitochondria. Nuclear DNA (nDNA) exists in large scale aggregate structures known as chromosomes which are composed of DNA wound up around bead-like proteins called histones. Whenever the cell needs to access the genetic information encoded in nDNA it will unravel the required section, read it, and then allow it to wind up once more in its protected conformation. In contrast, mitochondrial DNA (mtDNA) which is located inside mitochondria organelles, exists in single or multiple copies of a circular loop without any histone association.

Consequently, mtDNA is far more prone to damage than nDNA because it lacks the structural protection afforded by histone proteins. In addition, the highly oxidative environment inside mitochondria that exists due to the constant production of adenosine


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triphosphate (ATP) makes mtDNA even more prone to damage. Even though human mtDNA encodes only 13 proteins, a malfunctioning mitochondrion can activate apoptosis.

The types of molecules involved and the mechanism of repair that takes place is based on: 1. the type of damage on the DNA molecule 2. whether the cell has entered into a state of senescence 3. the phase of the cell cycle that the cell is in

SSiinnggllee ssttrraanndd aanndd ddoouubbllee ssttrraanndd DDNNAA ddaammaaggee

SSiinnggllee ssttrraanndd ddaammaaggee To repair damage to one of the two helical domains of DNA, the other strand must remain intact so that a replacement of damaged information can be made by the information from the undamaged copy. There are numerous mechanisms by which DNA repair can take place. These include

1. base excision repair (BER), which repairs damage due to alkylation or deamination;

2. nucleotide excision repair (NER), which repairs damage by UV light; and

3. mismatch repair (MMR), which corrects errors of DNA replication and recombination.

Cells that divide have an additional means of DNA repair via DNA polymerases. Cells that do not divide (such as brain and heart cells) cannot use this important DNA repair mechanism.

DDoouubbllee ssttrraanndd ddaammaaggee Most cells in the body have two copies of each chromosome, which becomes very useful during double strand damage. When damage occurs to both DNA strands, the only way that it can be repaired is by homologous recombination using the intact chromosome copy. This allows a damaged chromosome to be replaced, using the sister of the chromosome pair as the template.


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Poor DNA repair induces pathology

As cells get older the amount of DNA damage accumulates overtaking the rate of repair and resulting in a reduction of protein synthesis. As proteins in the cell are used for numerous vital functions the cell becomes slowly impaired and eventually dies. When enough cells in an organ reach such a state the organ itself will become compromised and the symptoms of disease begin to manifest. Experimental studies in animals, where genes associated with DNA repair were silenced, resulted in accelerated aging, early manifestation of age related diseases and increased susceptibility to cancer. In studies where the expression of certain DNA repair genes was increased resulted in extended lifespan and resistance to carcinogenic agents in cultured cells.

DNA repair rate is variable If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in senescence, apoptosis or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging (e.g. Werner's syndrome) and increased sensitivity to carcinogens (e.g Xeroderma Pigmentosum).

On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans (also known as "Conan the bacterium", listed in the Guinness Book of World Records as "the world's toughest bacterium"), exhibit remarkable resistance to lethal dosages of radioactivity, because their DNA repair enzymes are able to perform at


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unusually fast rates to keep up with radiation induced-damage, and because it carries 4–10 copies of the genome.

Studies in smokers have found that, for people with a mutation that causes them to express less of the powerful DNA repair gene , their vulnerability to lung and other smoking related cancers are increased. Single nucleotide polymorphisms (SNP) associated with this mutation can be clinically detected.

Hereditary DNA repair disorders Defects in the NER mechanism are responsible for several genetic disorders, including:

• xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer, incidence and premature aging

• Cockayne syndrome: hypersensitivity to UV and chemical agents • trichothiodystrophy: sensitive skin, brittle hair and nails

Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.

Other DNA repair disorders include:

• Werner's syndrome: premature aging and retarded growth • Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies

Chronic DNA repair disorders Chronic disease can be associated with increased DNA damage. For example, smoking cigarettes causes oxidative damage to the DNA and other components of heart and lung cells, resulting in the formation of DNA adducts (molecules that disrupt DNA). DNA damage has now been shown to be a causative factor in diseases from atherosclerosis to Alzheimer's, where patients have a lesser capacity for DNA repair in their brain cells. Mitochondrial DNA damage has also been implicated in numerous disorders.

Medicine & DNA repair modulation There is a vast body of evidence that has correlated DNA damage to death and disease. As indicated by new overexpression studies, increasing the activity of some DNA repair enzymes could decrease the rate of aging and disease. This may result in the development of human interventions that can add many healthy and disease-free years to an aging population. Not all DNA repair enzymes are beneficial when overexpressed, however. Some DNA repair enzymes can introduce new mutations in healthy DNA. Reduced substrate specificity has been implicated in these errors.

Cancer treatment Procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage and resulting in cell death. Cells that are most rapidly dividing such as cancer cells are preferentially affected. The side effect is that other non-cancerous but similarly rapidly dividing cells such as stem cells in the bone marrow are


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also affected. Modern cancer treatments attempt to localize the DNA damage to cells and tissues only associated with cancer.

Gene therapy For therapeutic uses of DNA repair, the challenge is to discover which particular DNA repair enzymes exhibit the most precise specificity for damaged sites, so its overexpression will lead to enhanced DNA repair function. Once the appropriate repair factors have been identified, the next step is in selecting the appropriate way to deliver them into cells, to generate viable disease and aging treatments. The development of smart genes, which are able to alter the amount of protein they produce based on changing cellular conditions, stand to increase the efficacy of DNA repair augmentation treatments.

Gene repair Unlike the multiple mechanisms of endogenous DNA repair, gene repair (or gene correction) refers to a form of gene therapy, which precisely targets and corrects chromosomal mutations responsible for a disorder. It does so by replacing the flawed DNA sequence with the desired sequence, using techniques such as oligonucleotide-directed mutagenesis. Genetic mutations requiring repair are normally inherited, but in some cases they can also be induced or acquired (such as in cancer).


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Lecture 3: RNA structure, transcription, post-transcriptional processing and drugs that inhibit these processes.

RNA Synthesis and Processing Major Classes of RNA

1. Messenger RNA: carries information from genes to ribosomes, where it is

translated into proteins.

In prokaryotic cells a. basic feature:

Most prokaryotic mRNA are poly cistronic. That is they carry the information for the production of multiple polypeptides.

b. Abundance mRNA accounts for only 5% of the total cellular RNA in prokaryotes

c. Stability Life time of prokaryotic mRNA is short, does not exceed more than a few minutes.

In eukaryotic cells a. basic feature:

Most prokaryotic mRNA are monocistronic. That is they carry the information for the production of a single polypeptide.

b. Abundance mRNA accounts for only 3% of the total cellular RNA. Its precursor hnRNA accounts for 7% of the cellular RNA in eukaryotes.

c. Stability Relatively stable and exhibites half-lives on the order of hours and days.

2. Ribosomal RNA: comprises approximately 50% of the mass of

ribosomes. The function of rRNA is both structural as well as catalytic. In prokaryotic cells

a. basic feature: There are three kinds of prokaryotic rRNA.

b. Abundance rRNA are the mostly abundant RNA class. They account for 80% of the total cellular RNA in prokaryotes.


the rRNA of eukaryotes are typically bigger than those of prokaryotes. Also , eukaryotes have four kinds of rRNA.

b. Abundance Approximately 4% of cellular rRNA is precursor rRNAm and 71% is fully processed rRNAs.


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3. Transfer RNA: serve to transfer amino acids to the ribosomes and

to facilitate the incorporation of the amino acids into newly synthesized proteins in a template-dependent manner. For each amino acid, there is one or more specific tRNA.

In prokaryotic cells a. basic feature:

tRNA are small in size with an average of 80 nucleotides. All tRNA have common structural features that allow them to function in the ribosomes.

b. Abundance The tRNA account for 15% of the total cellular RNA in prokaryotes.


eukaryotic tRNA are very similar to prokaryotic tRNA in size and structural features.

b. Abundance Same as in prokaryotes.

4. small RNAs (only in eukaryotes)


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Transcription The process of RNA synthesis directed by a DNA template is termed transcription, and occurs in three phases: initiation, elongation and termination. In transcription, DNA is copied to RNA by an enzyme called RNA polymerase . Transcription to yield an mRNA is the first step of protein biosynthesis .

1. initiation of transcription i. Promoter sequences. Unlike the initiation of replication, transcriptional

initiation does not require a primer. Promoter sequences are responsible for directing RNA polymerase to initiate transcription at a particular point. Promoter sequences differ between prokaryotes and eukaryotes

In genetics, a promoter is a DNA sequence that enables a gene to be transcribed. The promoter is recognized by RNA polymerase (RNAP), which then initiates transcription.

1. Prokaryotic promoters. The promoters for most prokaryotic genes have three sequence elements. a. Initiation site (startpoint). Transcription for most genes always starts

at the same base (position one). The startpoint is usually purine. b. Pribnow box. : lies 9-18 base pairs upstream of the startpoint.

i. Its either identical to or very similar to the sequence TATAAT. ii. The pribnow box also called -10 sequence because it is

usually found 10 bp upstream of the startpoint. c. The -35 sequence is a component of a typical prokaryotic promoter.

It is a TTGACA. Called -35 sequence because it is usually found 35bp upstream of the startpoint.

<--upstream downstream -->

2. Eukaryotic Promoters. Each type of eukaryotic RNA polymerase uses a different promoter. The promoters used by RNA polymerase I and II are similar to the prokaryotic promoter in that they are upstream of the startpoint. However, the promoters used by RNA polymerase III are unique because they are usually downstream of the startpoint.

ii. Initiation factors: 1. Prokaryotic σ factor is required for accurate initiation of transcription. 2. Eukaryotic initiation factors: the initiation of transcription in eukaryotes

is considerably more complex than in prokaryotes, partly because of the increased complexity of eukaryotic RNA polymerases and partly because of the diversity of their promoters.


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2. Elongation:

The basic requirement and fundamental mechanism of the elongation phase of RNA synthesis is the same in prokaryotes and eukaryotes. 1) Template: A single strand of DNA acts as a template to direct the formation of

complementary RNA during transcription. 2) Substrates: the four nucleosides triphosphates are needed as substrates for

RNA synthesis. 3) Direction of synthesis: RNA chain growth proceeds in the 5' to 3' direction. 4) Enzyme:

a. Prokaryotes have a single RNA polymerase responsible for all cellular synthesis.The structure of RNA polymerase is complex:

b. Eukaryotes have one mitochondrial and three nuclear RNA polymerase. The latter are distinct enzymes that function to synthesize different RNAs.

3. Termination: i. In prokaryotices:

There are two basic classes of termination event in prokaryotes

1. Intrinsic termination (Rho-independent termination) involves terminator sequences within the RNA as it is being made that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic DNA sequence that forms a hairpin.

2. Rho-dependent termination uses a termination factor called ρ factor to stop RNA synthesis at specific sites. When ρ-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription.


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ii. In eukaryotices: Very little is known about how they terminate transcription


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Action of antibiotics: Some antibiotics prevent bacterial cell growth by inhibiting RNA synthesis. For example, rifampin (useful in treatment of tuberculosis) inhibits the initiation of transcription by binding to the β-subunit of the prokaryotic RNA polymerase. , thus interfering with the formation of the first phosphdiester bond.

Dactinomycin "actinomycin D" (first antibiotic to find therapeutic application in tumor chemotherapy) binds to DNA template and interferes with the movement of RNA polymerase along the DNA.


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Posttranscriptional RNA processing Once a gene transcript has been synthesized, numerous post-

transcriptional modification or processing events may be needed

before the transcript is functional.

1. Prokaryotes: post-transcriptional processing of RNA is not as

extensive in prokaryotes as in eukaryotes; however, some

processing does occur.

2. Eukaryotes: Overall, post-transcriptional processing is more

extensive in eukaryotes than in prokaryotes. This partly is due to

the presence of a nucleus from which most RNAs must be

transported. RNAs are processed during this transport. Processing

gives them the characteristics they need to be functional in the

cytoplasm such as an increased stability of mRNAs as well as

allowing for another level of gene regulation.

a. The primary transcript (hnRNA) is capped at its 5' end as it is

being transcribed.

b. A poly (A) tail, 20 to 200 nucleotide in length is being added

to the 3' end of he transcript.

c. Splicing reactions remove introns and connect the exons.

(The most common cause of β-thalassemia are defects in mRNA splicing

of the β-globin gene. Mutations that affect the splicing create aberrant

transcript that are degraded before they are translated. If patients inherit

a single mutant gene thalassemia minor, the disease manifests itself

with a mild anemia. However, patents with homozygous mutations

thalassemia major have sever transfusion-dependent anemia.


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Lecture 4: Protein synthesis and translation in prokaryotic and eukaryotic cells and drugs that inhibit this process

Protein Synthesis: Protein biosynthesis is the process in which cells build proteins. The term is sometimes used to refer only to protein translation, but more often it refers to a multi-step process, beginning with transcription and ending with proteintranslation.


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Ribosome: A ribosome is an organelle composed of rRNA (synthesized in the nucleolus) and ribosomal proteins. It translates mRNA into a polypeptide chain (e.g., a protein). It can be thought of as a factory that builds a protein from a set of genetic instructions.

Free ribosomes Free ribosomes occur in all cells. Free ribosomes usually produce proteins that are used in the cytosol or in the organelle they occur in.

Membrane bound ribosomes When certain proteins are synthesized by a ribosome, it can become "membrane-bound", associated with the membrane of the nucleus and the rough endoplasmic reticulum (in eukaryotes only) for the time of synthesis. Translation (also called protein biosynthesis or polypeptide synthesis) is the second process in gene expression. In translation, messenger RNA is used as a template to produce a specific polypeptide according to the rules specified by the genetic code. Phases Translation proceeds in three phases: initiation, elongation, and termination (all describing the growth of the amino acid chain, or polypeptide that is the product of translation).

1. Initiation of translation involves the small ribosomal subunit binding to the 'start' codon on the mRNA, which indicates where the mRNA starts coding for the protein. This codon is most commonly an AUG. In eukaryotes amino acid encoded by the start codon is methionine. In bacteria, the protein starts instead with the modified amino acid N-formyl methionine (f-Met). In f-Met, the amino group has been blocked by a formyl group to form an amide, so this amino group can not form a peptide bond. This is not a problem because the f-Met is at the amino terminus of the protein.

2. The large subunit then forms a complex with the small subunit, and elongation proceeds. A new activated tRNA enters the A site of the ribosome and base pairs with the mRNA. The enzyme peptidyl transferase forms a peptide bond between the adjacent amino acids. As this happens, the amino acid on the P site leaves its tRNA and joins the tRNA at the A site. The ribosome then moves in relation to the mRNA shifting the tRNA at the A site on to the P whilst releasing the empty tRNA, this process is known as translocation.

3. This procedure repeats until the ribosome encounters one of three possible stop codons, where translation is terminated. This stalls protein growth, and release factors, proteins which mimic tRNA, enter the A site and release the protein in to the cytoplasm.

Synthesis of proteins can take place extremely quickly. This is aided by multiple ribosomes being able to attach themselves to one mRNA chain, thus allowing multiple proteins to be constructed at once. An mRNA chain with multiple ribosomes is called a polysome. Also, as prokaryotes have no nucleus, an mRNA can be translated while it is still being transcribed. This is not possible in eukaryotes as translation occurs in the cytoplasm, whereas transcription occurs in the nucleus.


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Protein Synthesis in Eukaryotes

A major difference between eukaryotes and prokaryotes is that, in a typical eukaryotic cell, protein synthesis takes place in the cytoplasm while transcription and RNA processing take place in the nucleus. In bacteria, these two processes can be coupled so that protein synthesis can start even before transcription has finished. INITIATION The cap-dependent translation initiation pathway Cap-dependent initiation is the major translation initiation pathway in eukaryotes

• eukaryotic mRNAs are monocistronic, capped at the 5' end and polyadenylated at the 3' end

• ribosomes dissociate into 40S and 60S subunits • 40S subunits locate the initiator AUG codon by scanning the mRNA from the

cap structure in the 3' direction for the first AUG codon • at the AUG codon the 60S ribosomal subunit joins the 40S initiation complex

to form an 80S ribosome competent for translation elongation:


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Fig.: Principle of cap-dependent translation initiation. AUU, stop codon; AAAn,

poly(A) tract. A large number of proteins, the eukaryotic translation initiation factors (eIF) catalyze individual steps in the pathway.

ELONGATION: The elongation in eukaryotes is very similar to that in prokaryotes. TERMINATION: Mechanism in eukaryotes is similar to that in prokaryotes

Drugs that inhibits protein synthesis 1. Erythromycin

Mechanism of Action Erythromycin inhibit protein synthesis by binding to the 23S rRNA molecule (in the 50S subunit) of the bacterial ribosome blocking the exit of the growing peptide chain. (Humans do not have 50 S ribosomal subunits, but have ribosomes composed of 40 S and 60 S subunits). Certain resistant microorganisms with mutational changes in components of this subunit of the ribosome fail to bind the drug. The association between erythromycin and the ribosome is reversible and takes place only when the 50 S subunit is free from tRNA molecules bearing nascent peptide chains. The non ionized from of the drug is considerably more permeable to cells, and this probably explains the increased antimicrobial activity that is observed in alkaline pH.


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2. Tetracyclines: Tetracyclines have the broadest spectrum of antimicrobial activity. Four fused 6-membered rings, as shown in the figure below, form the basic structure from which the various tetracyclines are made Mechanism of Action: Tetracyclines inhibit bacterial protein synthesis by blocking the attachment of the transfer RNA-amino acid to the ribosome. More precisely they are inhibitors of the codon-anticodon interaction. Tetracyclines can also inhibit protein synthesis in the host, but are less likely to reach the concentration required because eukaryotic cells do not have a tetracycline uptake mechanism. 3. Streptomycin: Streptomycin binds to

the 30S ribosome and changes its shape so that it and inhibits protein synthesis by causing a misreading of messenger RNA information.

4. Chloramphenicol: Chloromycetin is also a broad spectrum antibiotic that possesses activity similar to the tetracylines. At present, it is the only antibiotic prepared synthetically. It is reserved for treatment of serious infections because it is potentially highly toxic to bone marrow cells. It inhibits protein synthesis by attaching to the ribosome and interferes with the formation of peptide bonds between amino acids.


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Lecture 5: Post translational processes and protein folding

Post-Translational Modifications Some proteins must be modified in one or more of a number of ways before they realize their final functional form. The following are some of the modifications that have been found to occur to proteins after they have been synthesized:

1. Dealing with the N-terminal residue In bacteria, the N-terminal residue of the newly-synthesized protein is modified in bacteria to remove the formyl group. The N-terminal methionine may also be removed. In eukaryotes, the methionine is also subject to removal.

2. Amino Acid Modifications a. Acetylation b. Phosphorylation c. Methylation d. Carboxylation e. Hydroxylation f. Glycosylation g. Nucleotidylation h. Lipid Addition

Others The protein, thyroglobin, is iodinated during the synthesis of thyroxine.

i. Adding Prosthetic Groups Proteins that require a prosthetic group for activity must have this group added. For example, the haem (heme) group must be added to globins and cytochromes; Fe-S clusters must be added to ferredoxins.

j. Forming Disulfide Bonds Many extracellular proteins contain disulfide cross-links (intracellular proteins almost never do). The cross-links can only be established after the protein has folded up into the correct shape.

Proteolytic Processing

Some proteins are synthesized as inactive precursor polypeptides which become activated only after proteolytic cleavage of the precursor polypeptide chain. Two well-known examples are:

Chymotrypsin & Trypsin

Chymotrypsin and trypsin are both synthesized as zymogens. Cleavage of chymotrypsinogen between Arg15 and Ile 16 by trypsin yields the enzymatically active pi-chymotrypsin. Two further proteolytic cleavages catalyzed by chymotrypsin removes the dipeptides Ser14-Arg15 and Thr147-Asn148 to yield alpha-chymotrypsin. Trypsin is activated by the removal of the N-terminal seven amino acids. Insulin Insulin is synthesized as a precursor polypeptide. The initial preproinsulin contains a signal sequence since the protein is targeted for secretion.


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Protein Folding

As they are being synthesized, proteins must adopt the correct conformation for their function. Protein folding is the process by which a string of amino acids (the chemical building blocks of protein) interacts with itself to form a stable three- dimensional structure during production of the protein within the cell. The process is roughly analogous to the ways in which a length of wire may be twisted onto or against itself to form various functional entities, for example a spring, a paperclip or a coat hanger. Folding occurs very rapidly, probably within milliseconds of production of the string of amino acids, and results in 3-D conformations which usually are quite stable, with specific biological functions. The folding of proteins thus facilitates the production of discrete functional entities, including enzymes and structural proteins, which allow the various processes associated with life to occur. Importantly, folding not only

1. allows the production of structures which can perform particular functions in the cellular milieu, but also

2. it prevents inappropriate interactions between proteins, in that folding hides elements of the amino acid sequence which if exposed would react non-specifically with other proteins.

Proteins may either fold spontaneously or they may need the assistance of chaperone proteins so that they do not get trapped in stable folding intermediates but rather fold into the correct final conformation. There are 3 major classes of chaperones:

• The Hsp70 family • The Hsp 60 family

Protein misfolding diseases In many cases, misfolded proteins are recognized to be undesirable by a group of proteins called heat shock proteins, and consequently directed to protein degradation machinery in the cell. This involves conjugation to the protein ubiquitin, which acts as a tag that directs the proteins to proteasomes, where they are degraded into their constituent amino acids. Hence many protein misfolding diseases are characterized by absence of a key protein, as it has been recognised as dysfunctional and eliminated by the cell’s own machinery. Diseases caused by lack of a particular functioning protein, due to its degradation as a consequence of misfolding, include:

• cystic fibrosis (misfolded CFTR protein), • Marfan syndrome (misfolded fibrillin), • Fabry disease (misfolded alpha galactosidase), • Gaucher’s disease (misfolded beta glucocerebrosidase) and • retinitis pigmentosa 3 (misfolded rhodopsin).


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In addition, some cancers may be associated with misfolding, and hence ineffective functioning, of tumour suppressor proteins such as p53. Many protein misfolding diseases are characterized not by disappearance of a protein but by its deposition in insoluble aggregates within the cell. Diseases caused by protein aggregation include:

• Alzheimer’s disease (deposits of amyloid beta and tau), • Type II diabetes (depositis of amylin), • Parkinson’s disease (deposits of alpha synuclein), and • the spongiform encephalopathies such as Creutzfeldt-Jakob disease

(deposits of prion protein). Protein misfolding appears at least in some cases to be due to mutations (missing or incorrect amino acids) in the protein which destabilise it such that it is more likely to fold incorrectly. Alternatively, the misfolding could occur due to progressively lower levels of chaperone proteins in ageing neurons. It may also be that mutations or other changes in the chaperone proteins themselves cause them to actually promote misfolding, rather than guard against it.


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Lecture 6: Protein targeting and degradation

Protein Targeting Proteins that must be targeted within the cell must be intercepted early during synthesis so that this can happen correctly. As a protein is being synthesized, decisions must be taken about sending it to the correct location in the cell where it will be required. The information for doing this resides in the nascent protein sequence itself. Once the protein has reached its final destination, this information may be removed by proteolytic processing.

Targeting in Bacteria In bacterial cells, the targeting decision is relatively straightforward: is the protein destined to be an intracellular protein or an extracellular one? Secreted proteins contain a signal sequence. This is a short (6 - 30) stretch of hydrophobic amino acids, flanked on the N-terminal side by one or more positively charged amino acids such as lysine or arginine, and containing neutral amino acids with short side-chains (such as glycine or alanine) at the cleavage site. As proteins with signal sequences are synthesized, they are bound by the SecB protein. This prevents the protein from folding. SecB delivers the protein to the cell membrane where is secreted through a pore formed by the SecE and SecY proteins. Secretion is driven by the SecA ATPase. After the protein has been secreted, the signal sequence is removed by a membrane bound leader peptidase.


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Targeting in Eukaryotes Targeting in eukaryotes is necessarily more complex due to the multitude of internal compartments: Proteins that are synthesized on free ribosomes may also be targeted within the cell:

• Proteins that are targeted for organelles have their own N-terminal uptake-targeting sequence(s) that determines whether the protein must cross one or two membranes. In the former case, proteins destined for the intermembrane space of the mitochondrion are first transported into the matrix (mitochondrion) and then re-transported back through the inner mitochondrial membrane to the intermembrane space.

Proteins that must be targeted to the nucleus have a nuclear localization signal (NLS). Once common type of signal is a series of five or so closely spaced positively charged amino acids.

The Signal Sequence hypothesis was first enunciated by Gunther Blöbel who was awarded the Nobel Prize in Medicine in 1999 for his work.


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The following diagram summarizes the choices/fates available to newly synthesized proteins in a eukaryotic cell:

The SRP cycle The signal recognition particle (SRP) associates with ribosomes that are in the process of translating the mRNA for a secretory protein. The protein has a signal at the N-terminus. Subsequently, the ribosome-bound SRP interacts with the SRP-receptor a component of the ER membrane. Finally, SRP recycles to associate with another ribosome, and translation continues with the secretory protein transversing the membrane through a channel called the translocon.


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Targeted Protein Degradation In order to keep a cell working it needs to remove:

1. incorrectly synthesized proteins (with errors in amino acid sequence) 2. damaged proteins (i.e. oxidative damage) 3. cell-cycle specific proteins 4. other signaling proteins which are no longer necessary

One mechanism of protein degradation is via lysosomes.

Lysosomes are acidic vesicles that contain about 50 different enzymes involved in degradation:

1. proteases (cathepsins): cleave peptide bonds 2. phosphatases: remove covalently bound phosphates 3. nucleases: cleave DNA/RNA 4. lipases: cleave lipid molecules 5. carbohydrate-cleaving enzymes: remove covalently bound sugars from

glycoproteins Lysosomes often secrete their contents into the extracellular medium via exocytosis. Lysosomes can also target damaged organelles in a process called autophagy. Sometimes, lysosomes are triggered to rupture inside a cell, resulting in autolysis, also called apoptosis or programmed cell death.

Another major mechanism is via ubiquitin labeling of surplus proteins:

• Ubiquitin (a small 76-residue protein) is attached to the protein: o First, an activating enzyme attaches itself to the carboxy terminus of

free ubiquitin in an ATP-dependent process. o Then, the activated ubiquitin is transferred onto a second enzyme

which at the same time recognizes damaged proteins. o The activated ubiquitin is then covalently linked to lysine residues on

the surface of the damaged protein.

• These ubiquitin-tagged proteins are now recognized by specific proteases in the cytosol which in turn cleave and degrade the tagged protein.

• These proteases are combined in a very large protein complex called the proteasome.


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47

Lecture 7: Biochemistry of cancer and tumor markers

Biochemistry of Cancer and Tumor Markers

Cancer is a long term multistage genetic process. The first stage is when the DNA is

damaged by some form of carcinogen: physical, chemical, and biologic agents (e.g.

smoking, radiation, chemicals, and virus). These agents damage or alter DNA, so

that cancer is truly a disease of the genome. At some later time, additional damage

occurs that eventually leads to chromosome breakdown and rearrangement. This

process produces a new phenotype that loses control over the process of mitosis.

The process of mitosis continues and unlimitedly produces malignant tumor cells.

Eventually, there is a production of a growing mutant cell that expresses oncogenes.

(Oncogenes: are genes capable of inducing or maintaining transformation of cells).

Benign tumor cells have lost growth control but do not metastasize.

Much current interest in cancer is focused on the study of oncogenes and tumor

suppressor genes. Normal cells contain potential precursors of oncogenes,

designated proto-oncogenes. Activation of these genes to oncogenes is achieved by

at least five mechanisms:

1. promoter and

2. enhancer insertion

3. Chromosomal translocation,

4. gene amplification

5. Point mutation.

Activated oncogenes influence cellular growth by perturbing normal cellular

mechanism of growth control, by acting as growth factors or receptors, and probably

by other means as well.

Tumor suppressor genes are now recognized as key players in the genesis of

cancer.

Important tumor suppressor genes include RB1 and P53, both of which are nuclear

phosphoproteins and probably affect the transcription of genes involved in regulating

events in the cell cycle.

Tumor progression reflects instability of the tumor genome probably due at least in

part to defects in DNA repair systems, activation of additional oncogenes, and

inactivation of additional tumor suppressor genes.


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The extensive biochemical analyses of the Morris minimal-deviation

Hepatomas (tumors originally induced in rats by feeding them the carcinogens

fluorenylphthalamic acid, fluorenylacetamide compounds, or trimethylaniline. These hepatocellular carcinomas are transplantable in an inbred host strain

of rats and have a variety of growth rates and degrees of differentiation. All these tumors are

malignant and eventually kill the host. The term “minimal deviation” was coined by Potter to convey the idea that some of these neoplasms differ only slightly from normal hepatic

parenchymal cells) led Weber to formulate the “molecular correlation concept” of

cancer, which states that “the biochemical strategy of the genome in neoplasia

could be identified by elucidation of the pattern of gene expression as revealed in the activity, concentration, and isozyme aspects of key enzymes and their

linking with neoplastic transformation and progression.”

Weber proposed three general types of biochemical alterations associated with

malignancy:

1. transformation-linked alterations that correlate with the events of malignant

transformation and that are probably altered in the same direction in all malignant

cells;

2. progression-linked alterations that correlate with tumor growth rate, invasiveness,

and metastatic potential; and

3. coincidental alterations that are secondary events and do not correlate strictly

with transformation or progression.

Those metabolic pathways that contained enzymes which fulfilled one or more of

these criteria are indicated in Table (1) along with the alteration that was observed in

cancer. Table(1) Molecular Correlation Concept and Affected Processes Biochemical Process Alteration in Cancer Cells Pyrimidine and purine synthesis Increased

Pyrimidine and purine catabolism Decreased

RNA and DNA synthesis Increased

Glucose catabolism Increased

Glucose synthesis Decreased

Amino acid catabolism (for gluconeogenesis) Decreased

Urea cycle Decreased


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Enzymes in Malignancy Plasma total enzyme activities may be raised or an abnormal isoenzyme detected,

in several neoplastic disorders.

• Serum prostatic (tartrate-labi!e) acid phosphatase activity rises in some cases of

malignancy of the prostate gland.

• Any malignancy may be associated with a non-specific increase in plasma LD1

(HBD) and. occasionally, transaminase activity.

• Plasma transaminase and alkaline phosphatase estimations may be of value to

monitor treatment of malignant disease. Raised levels may indicate secondary

deposits in liver or of alkaline phosphatase, in bone. Liver deposits may also cause

an increase in plasma LD or GGT.

• Tumors occasionally produce a number of enzymes, such as the 'Regan' ALP

isoenzyme.' LD (HBD) or CK-BB. assays of which may be used as an aid to

diagnosis or for monitoring treatment.

A number of oncodevelopmental tumor-associated antigens appear on tumor cells as

a result of the apparent re-expression (or increased expression) of embryonic genes,

and a number of these are useful as tumor markers for cancer diagnosis and disease

progression.

These include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), and a

number of inappropriately (ectopically) produced hormones. (Table (2).


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Classification of Tumor Markers

Tumor markers come from a variety of groups:

Enzymes, glycoproteins, hormones and hormone-like substances, hormone

receptors, oneogenes, and oneogene reseptors. The list of tumor markers that arise

from this list is quite extensive. However, because of the low sensitivity and

specificity of most tumor markers, the Food and Drug Administration (FDA) has

approved only a few assay kits as tumor markers.

What are they?

Tumor markers are substances, usually proteins that are produced by the body in

response to cancer growth or by the cancer tissue itself. Some tumor markers are

specific, while others are seen in several cancer types. Many of the well-known

markers are also seen in non-cancerous conditions. Consequently, these tumor

markers are not diagnostic for cancer.

There are only a handful of well-established tumor markers that are being routinely

used by physicians. Many other potential markers are still being researched. Some

marker tests cause great excitement when they are first discovered but, upon further

investigation, prove to be no more useful than markers already in use.

The goal is to be able to screen for and diagnose cancer early, when it is the most

treatable and before it has had a chance to grow and spread. So far, the only tumor

marker to gain wide acceptance as a general screen is the Prostate Specific Antigen

(PSA) for men. Other markers are either not specific enough (too many false

positives, leading to expensive and unnecessary follow-up testing) or they are not

elevated early enough in the disease process.

Some people are at a higher risk for particular cancers because they have inherited a

genetic mutation. While not considered tumor makers, there are tests that look for

these mutations in order to estimate the risk of developing a particular type of cancer.

BRCA1 and BRCA2 are examples of gene mutations related to an inherited risk of

breast cancer and ovarian cancer.


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Why are they done? Tumor markers are not diagnostic in themselves. A definitive diagnosis of cancer is

made by looking at biopsy specimens (e.g., of tissue) under a microscope. However,

tumor markers provide information that can be used to:

Screen: Most markers are not suited for general screening, but some may be used in

those with a strong family history of a particular cancer. In the case of genetic

markers, they may be used to help predict risk in family members. (PSA testing for

prostate cancer is an example).

Help diagnose: In a patient that has symptoms, tumor markers may be used to help

identify the source of the cancer, such as CA-125 for ovarian cancer, and to help

differentiate it from other conditions.

Stage: If a patient does have cancer, tumor marker elevations can be used to help

determine how far the cancer has spread into other tissues and organs.

Determine prognosis. Some tumor markers can be used to help doctors determine

how aggressive a cancer is likely to be.

Guide Treatment. Some tumor markers will give doctors information about what

treatments their patients may respond to.

Monitor Treatment. Tumor markers can be used to monitor the effectiveness of

treatment, especially in advanced cancers. If the marker level drops, the treatment is

working; if it stays elevated, adjustments are needed.

Determine recurrence. Currently, one of the biggest uses for tumor markers is to

monitor for cancer recurrence. If a tumor marker is elevated before treatment, low

after treatment, and then begins to rise over time, then it is likely that the cancer is

returning. (If it remains elevated after surgery, then chances are that not all of the

cancer was removed.)


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53

Lecture 8: The biochemistry of nucleic acids metabolism

Nucleic acids Metabolism

Synthesis of purine nucleotides The atoms of the purine ring are contributed by a number of compounds, including amino acids (aspartic acid, glycine, and glutamine), CO2 and N10-formyltetrahydrofolate.

Sources of the individual atoms in the purine ring

A. Synthesis of 5-phosphoribosyl-1-pyrophosphate(PRPP) PRPP is an "activated pentose" that participates in the synthesis of purines and pyrimidines and the salvage of purine bases. Synthesis of PRPP from ATP and ribose 5-phosphate is catalyzed by PRPP synthetase as shown in the figure bellow:

The enzyme is activated by inorganic phosphate (Pi) and inhibited by purine nucleotides (end-product inhibition).

OH

OH OH

Ophosphate-OH2C

Ribose 5-phosphate

O

OH OH

Ophosphate-OH2C

Ribose 5-phosphate

-phosphate-phoshate

ActivatorPi

PRPP synthetase

inhibitorspurine ribonucleotides

ATP AMP

Mg+2


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Note: the sugar moiety of PRPP is ribose and therefore ribonucleotieds are the end products of de novo purine synthesis. When deoxyribonucleotides are required for DNA synthesis, the ribose sugar moiety is reduced.

B. Synthesis of 5'-phosphoribosylamine Synthesis of 5'-phosphoribosylamine from PRPP and glutamine is shown:

The amide group of glutamine replaces the pyrophosphate group attached to carbon 1 of PRPP. The enzyme glutamine:phosphoribosyl pyrophosphate amidotransferase, is inhibited by the purine 5'-nucleotide


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AMP, GMP, and IMP- the end products of the pathway. This is the committed step in purine nucleotide biosynthesis. The rate of the reaction is also controlled by the intracellular concentration of the substrates glutamine and PRPP. C. Synthesis of inosine monophosphate, the "parent" purine nucleutide The next nine steps in purine nuclutide biosynthesis leading to the synthsis of IMP (whose base is hypoxanthine) is illustrated bellow:


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PABA analogs • Sulfonamides are structural analogs of PABA that competitively inhibit bacterial synthesis of folic acid . Because purine synthesis requires THF as a coenzyme, the sulfa drugs slow down this pathway in bacteria. • Humans cannot synthesize folic acid, and must rely on external sources of this vitamin. Therefore, sulfa drugs do not interfere with human purine synthesis.

Folic acid analogs • Methotrxate and related compounds inhibit the reduction of dihydrofolate to tetrahydrofolate, catalyzed by dihydrofolate reductase. • These drugs limit the amount of tetrahydrofolate available for use in purine synthesis and thus slow down DNA replication in mammalian cells. These compounds are therefore useful in treating rapidly growing cancers, but are also toxic to all dividing cells.

This pathway requires four ATP molecules as an energy source. Two steps in the pathway require N10-formyltetrahydrofolate. D. Synthetic inhibitors of purine synthesis Some synthetic inhibitors of purine synthesis (for example , the sulfonamides), are designed to inhibit the growth of rapily dividing microorganisms without interfering with human cell functions. Other purine synthesis inhibitors such as structural analogs of folic acid (for example, methotrxate are used pharmacologically to control the spred of cancer by interfering with the synthesis of nucleotides and therefore of DNA and RNA as shown in the figure above. Note: Inhibitores of human purine synthesis are extremely toxic to tissues, especially to developing structures such as in a fetus or to cell types that normaly replicate rapidly including those of bone marrow, skin, gastrointestinal tract, immune system or hair follicles. As a result individuals taking such anti-cancer drugs can experience adverse effects, including anemia, scaly skin, GI tract disturbance, immunodeficiencies and baldness. Trimethoprim another folate analog has a potent antbacterial activity because of its selective inhibition of bacterial dihydrofolate reductase. (see the attached figure)


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E. Conversion of IMP to AMP and GMP • The conversion of IMP to either AMP or GMP uses a two-step

energy requiring pathway.

• The synthesis of AMP requires GTP as an energy source, whereas the synthesis of GMP requires ATP.

• The first reaction in each pathway is inhibited by the end product of that pathway. This provides a mechanism for diverting IMP to the synthesis of the species of purine present in lesser amounts.

• If both AMP and GMP are present in adequate amounts, the de novo pathway of purine synthesis is turned off at the aminotransferase step.

• Mycophenolic acid (MPA) is a potent reversible, uncompetitive inhibitor of IMP dehydrogenises that is being used successfully in preventing graft rejection. It blocks the de novo formation of GMP, thus depriving rapidly proliferating cells including T and B cells of a key component of nucleic acids.

• Graft: any organ, tissue or object used for transplantation to replace a faulty part of the body.


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F. Conversion of nucleoside monophosphate to nucleoside diphosphates and triphosphates

Nucleoside diphosphates (NDP) are synthesized from the corresponding nucleoside monophosphate (NMP) by base-specific nucleoside monophosphate kinases. These kinases do not discriminate between ribose or deoxyribose in the substrate. Adenylate kinase is particularly active in the liver and muscle where the turnover of energy from ATP is high. Nucloside diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase-an enzyme that unlike mono-phosphate kinases has broad specificity.


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G. Catabolism and Salvage of Purine Nucleotides

The final product of purine degradation is uric acid, which is produced via the following pathway: a. Hypoxanthine, from the breakdown of AMP, is oxidized to xanthine

by the enzyme xanthine oxidase. b. Guanine, from the breakdown of GMP, is deaminated to xanthine. c. Xanthine is oxidized to uric acid by xanthine oxidase.

1. Oxygen (O2) is required, and hydrogen peroxide (H2O2) is generated in the oxidations by xanthine oxidase.

2. Xanthine oxidase contains molybdenum, which is why this element is required in trace amounts in humane. This enzyme also contains iron and sulfur.

If this process is occurring in tissues other than liver, most of the ammonia will be transported to the liver as glutamine for ultimate excretion as urea.

Xanthine, like hypoxanthine, is oxidized by oxygen and xanthine oxidase with the production of hydrogen peroxide. In man, the urate is excreted and the hydrogen peroxide is degraded by catalase. Xanthine oxidase is present in significant concentration only in liver and intestine. The


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pathway to the nucleosides, possibly to the free bases, is present in many tissues.

Catabolism of the purine nucleotides leads ultimately to the production of uric acid which is insoluble and is excreted in the urine as sodium urate crystals

The synthesis of nucleotides from the purine bases and purine nucleosides takes place in a series of steps known as the salvage pathways. The free purine bases---adenine, guanine, and hypoxanthine---can be reconverted to their corresponding nucleotides by phosphoribosylation. Two key transferase enzymes are involved in the salvage of purines: adenosine phosphoribosyltransferase (APRT), which catalyzes the following reaction:

adenine + PRPP <-----> AMP + PPi

and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the following reactions:

hypoxanthine + PRPP <------> IMP + PPi

guannine + PRPP <--------> GMP + PPi

Purine nucleotide phosphorylases can also contribute to the salvage of the bases through a reversal of the catabolism pathways. However, this pathway is less significant than those catalyzed by the phosphoribosyltransferases.

Clinical Significances of Purine Metabolism Clinical problems associated with nucleotide metabolism in humans are predominantly the result of abnormal catabolism of the purines. The clinical consequences of abnormal purine metabolism range from mild to severe and even fatal disorders. Clinical manifestations of abnormal purine catabolism arise from the insolubility of the degradation byproduct, uric acid. Excess accumulation of uric acid leads to hyperuricemia, more commonly known as gout. This condition results from the precipitation of sodium urate crystals in the synovial fluid of the joints, leading to severe inflammation and arthritis.

Most forms of gout are the result of excess purine or of a partial deficiency in the salvage enzyme, HGPRT. Most forms of gout can be treated by administering the antimetabolite: allopurinol. This compound is a structural analog of hypoxanthine that strongly inhibits xanthine oxidase.


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Two severe disorders, both quite well described, are associated with defects in purine metabolism:

1. Lesch-Nyhan syndrome: Lesch-Nyhan syndrome results from the loss of a functional HGPRT gene. Patients with this defect exhibit not only severe symptoms of gout but also a severe malfunction of the nervous system. In the most serious cases, patients resort to self-mutilation. Death usually occurs before patients reach their 20th year.

2. Severe combined immunodeficiency disease (SCID): SCID is

caused by a deficiency in the enzyme adenosine deaminase (ADA). This is the enzyme responsible for converting adenosine to inosine in the catabolism of the purines. This deficiency selectively leads to a destruction of B and T lymphocytes, the cells that mount immune responses. In the absence of ADA, deoxyadenosine is phosphorylated to yield levels of dATP that are 50-fold higher than normal. The levels are especially high in lymphocytes, which have abundant amounts of the salvage enzymes, including nucleoside kinases. High concentrations of dATP inhibit ribonucleotide reductase, thereby preventing other dNTPs from being produced. The net effect is to inhibit DNA synthesis. Since lymphocytes must be able to proliferate dramatically in response to antigenic challenge, the inability to synthesize DNA seriously impairs the immune responses, and the disease is usually fatal in infancy unless special protective measures are taken. A less severe immunodeficiency results when there is a lack of purine nucleoside phosphorylase (PNP), another purine-degradative enzyme.

One of the many glycogen storage diseases von Gierke's disease also leads to excessive uric acid production. This disorder results from a deficiency in glucose 6-phosphatase activity. The increased availability of glucose-6-phosphate increases the rate of flux through the pentose phosphate pathway, yielding an elevation in the level of ribose-5-phosphate and consequently PRPP. The increases in PRPP then result in excess purine biosynthesis.


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Disorders of Purine Metabolism

Disorder Defect Nature of Defect Comments

Gout PRPP synthetase

increased enzyme activity due to elevated Vmax

hyperuricemia

Gout PRPP synthetase enzyme is resistant to feed-back inhibition

hyperuricemia

Gout PRPP synthetase

enzyme has increased affinity for ribose-5-phosphate (lowered Km)

hyperuricemia

Gout PRPP amidotransferase

loss of feed-back inhibition of enzyme

hyperuricemia

Gout HGPRTa partially defective enzyme hyperuricemia

Lesch-Nyhan syndrome HGPRT lack of enzyme see above

SCID ADAb lack of enzyme see above

Immunodeficiency PNPc lack of enzyme see above

Renal lithiasis APRTd lack of enzyme 2,8-dihydroxyadenine renal lithiasis

Xanthinuria Xanthine oxidase lack of enzyme hypouricemia and xanthine renal lithiasis

von Gierke's disease</TD

Glucose-6-phosphatase

enzyme deficiency see above

aHypoxanthine-guanine phosphoribosyltransferase; badenosine deaminase; cpurine nucleotide phosphorylase; dadenosine phosphoribosyltransferase


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PPyyrriimmiiddiinnee NNuucclleeoottiiddee BBiioossyynntthheessiiss Synthesis of the pyrimidines is less complex than that of the purines, since the base is much simpler.

A. Synthesis of carbamoyl phosphate The carbamoyl phosphate used for pyrimidine nucleotide

synthesis is derived from glutamine and bicarbonate, within the cytosol, as opposed to the urea cycle carbamoyl phosphate derived from ammonia and bicarbonate in the mitochondrion. The urea cycle reaction is catalyzed by carbamoyl phosphate synthetase I (CPS-I) whereas the pyrimidine nucleotide precursor is synthesized by CPS-II.

2ATP + CO2+ Glutamine

2ADP +PiGlutamate

Carbomylphosphatesynthetase II

Regulation of Pyrimidine Synthesis- In mammalian cells, CPS II is inhibited by UTP and activated byATP and PRPP

P

O

O-O

OH

H2N

O

carbamoyl phosphate


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B. Synthesis of orotic acid Carbamoyl phosphate is then condensed with aspartate in a

reaction catalyzed by the rate limiting enzyme of pyrimidine

nucleotide biosynthesis, aspartate transcarbamoylase (ATCase),

forming carbomyl aspartate . the pirimidine ring is then closed

hydrolytically by dihydroorotase. The resulting dihydroorotate is

oxidized to produce orotic acid . the enzyme that produce orotate

(dihydroorotate dehydrogenase) is located inside the mitochondia.

All other reactions in the pyrimidine biosynthesis are cytosolic.

Carbamoyl phosphate

Aspartate Pi

Aspartate transcarbamoylase

NH

O

-O

OHO

NH2

O

Carbamoyl aspartate

Regulation of Pyrimidine Synthesis- In prokaryotic cells, aspartate transcarbamoylase is inhibited by CTP and is the regulated step

HN

O

NH

O

O

-O

Dihydroorotate

HN

O

NH

O

O

-O

Orotate

NAD+NADH +H+

H2O

Dihydroorotase

Dihydroorotasedehydrogenase


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C. Formation of a pyrimidine nucleotide

The completed pyrimidine ring is converted to the nucleotide

orotidine 5'-monophosphate (OMP) in the second stage of

pyrimidine nucleotide synthesis. PRPP is again the ribose 5-

phosphate donor. The enzyme orotate phosphoribosyltransferase

produces OMP and releases pyrophosphate, thereby making the

reaction biologically irreversible.

HOOH

ON

ONH

O

OHOP

O

O

HO

HO

Orotidine 5'-monophosphate OMP

PRPP PPiHNO

NH

OO

-O

Orotate

OMP decraboxylase

HOOH

ON

O

NH

OP

O

O

HO

HO

uridine 5'-monophosphate UMP

Orotic Aciduria-Low activities of orotidine phosphate decarboxylase and orotate phosphoribosyltransferas result in abnormal growth, megaloblastic anemia and the excretion of large amounts of orotate in the urine.-Feeding a diet rich in uridine results in improvement of the anemia and decreased excretion of orotate CO2


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Note: both purine and pyrimidine synthesis thus require glutmine

and PRPP as essential precuersors.

The synthesis of pyrimidines differs in two significant ways

from that of purines.

1. First, the ring structure is assembled as a free base, not built

upon PRPP. PRPP is added to the first fully formed pyrimidine

base (orotic acid), forming orotate monophosphate (OMP),

which is subsequently decarboxylated to UMP.

2. Second, there is no branch in the pyrimidine synthesis pathway.

D. Synthesis of uridine triphosphate and cytidine triphosphate

UMP is phosphorylated twice to yield UTP (ATP is the phosphate

donor). The first phosphorylation is catalyzed by uridylate kinase

and the second by ubiquitous nucleoside diphosphate kinase.

Finally UTP is aminated by the action of CTP synthase, generating

CTP.


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E. Synthesis of Thymine Nucleotides The thymine nucleotides are in turn derived by de novo synthesis

from dUMP or by salvage pathways from deoxyuridine or

deoxythymidine.

The de novo pathway to dTTP synthesis first requires the use of

dUMP from the metabolism of either UDP or CDP. The dUMP is

converted to dTMP by the action of thymidylate synthase. The

methyl group (recall that thymine is 5-methyl uracil) is donated by

tetrahydrofolate, similarly to the donation of methyl groups during

the biosynthesis of the purines.

The salvage pathway to dTTP synthesis involves the enzyme

thymidine kinase which can use either thymidine or deoxyuridine

as substrate:

The activity of thymidine kinase (one of the various

deoxyribonucleotide kinases) is unique in that it fluctuates with

the cell cycle, rising to peak activity during the phase of DNA

synthesis; it is inhibited by dTTP.

F. Clinical Relevance of Tetrahydrofolate Tetrahydrofolate (THF) is regenerated from the dihydrofolate

(DHF) product of the thymidylate synthase reaction by the action

of dihydrofolate reductase (DHFR), an enzyme that requires

NADPH. Cells that are unable to regenerate THF suffer defective

DNA synthesis and eventual death. For this reason, as well as the

fact that dTTP is utilized only in DNA, it is therapeutically possible

to target rapidly proliferating cells over non-proliferating cells


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through the inhibition of thymidylate synthase. Many anti-cancer

drugs act directly to inhibit thymidylate synthase, or indirectly, by

inhibiting DHFR.

The class of molecules used to inhibit thymidylate synthase is

called the suicide substrates, because they irreversibly inhibit the

enzyme. Molecules of this class include 5-fluorouracil and 5-

fluorodeoxyuridine. Both are converted within cells to 5-

fluorodeoxyuridylate, FdUMP. It is this drug metabolite that

inhibits thymidylate synthase. Many DHFR inhibitors have been

synthesized, including methotrexate, and trimethoprim. Each of

these is an analog of folic acid.

G. Salvage of Pyrimidine Nucleotide The salvage of pyrimidine bases has less clinical significance

than that of the purines, owing to the solubility of the by-products

of pyrimidine catabolism. Uridine and cytidine can be salvaged by

uridine-cytidine kinase.

Deoxycytidine can be salvaged by deoxycytidine kinase and

thymidine can be salvaged by thymidine kinase. Each of these

enzymes catalyzes the phosphorelation of nucleotides utilizing

ATP and forming UMP, CMP, dCMP and TMP

H. Catabolism of Pyrimidine Nucleotide Catabolism of the pyrimidine nucleotides leads ultimately to β-

alanine (when CMP and UMP are degraded) or β-aminoisobutyrate


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(when dTMP is degraded) {which can serve as precursors of acetyl

CoA and succinyl CoA respeciely},and NH3 and CO2.

Clinical Significances of Pyrimidine Metabolism Because the products of pyrimidine catabolism are soluble,

few disorders result from excess levels of their synthesis or

catabolism. Two inherited disorders affecting pyrimidine

biosynthesis are the result of deficiencies in the bifunctional

enzyme catalyzing the last two steps of UMP synthesis, orotate

phosphoribosyl transferase and OMP decarboxylase. These

deficiencies result in orotic aciduria that causes retarded growth,

and severe anemia. Leukopenia is also common in orotic

acidurias. The disorders can be treated with uridine and/or

cytidine, which leads to increased UMP production via the action

of nucleoside kinases. The UMP then inhibits CPS-II, thus

attenuating orotic acid production.


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Formation of Deoxyribonucleotides The typical cell contains 5 to10 times as much RNA (mRNAs,

rRNAs and tRNAs) as DNA. Therefore, the majority of nucleotide

biosynthesis has as its purpose the production of rNTPs. However,

because proliferating cells need to replicate their genomes, the

production of dNTPs is also necessary.

De novo synthesis and most of the salvage pathways involve the

ribonucleotides. (Exception is the small amount of salvage of

thymine indicated above.) Deoxyribonucleotides for DNA synthesis

are formed from the ribonucleotide diphosphates (in mammals

and E. coli).

A base diphosphate (BDP) is reduced at the 2' position of the

ribose portion using the protein, thioredoxin and the enzyme

nucleoside diphosphate reductase. Thioredoxin has two sulfhydryl

groups which are oxidized to a disulfide bond during the process.

In order to restore the thioredoxin to its reduced for so that it can

be reused, thioredoxin reductase and NADPH are required.


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This system is very tightly controlled by a variety of allosteric

effectors. dATP is a general inhibitor for all substrates and ATP an

activator. Each substrate then has a specific positive effector (a

BTP or dBTP). The result is a maintenance of an appropriate

balance of the deoxynucleotides for DNA synthesis.

Ribonucleotide reductase (RR) is a multifunctional enzyme that

contains redox-active thiol groups for the transfer of electrons

during the reduction reactions. In the process of reducing the

rNDP to a dNDP, RR becomes oxidized. RR is reduced in turn, by

either thioredoxin or glutaredoxin. The ultimate source of the

electrons is NADPH. The electrons are shuttled through a complex

series of steps involving enzymes that regenerate the reduced


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forms of thioredoxin or glutaredoxin. These enzymes are

thioredoxin reductase and glutathione reductase respectively.

SUMMARY

1. Ingested nucleic acids are degraded to purines and pyrimidines. New purines and

pyrimidines are formed from amphibolic intermediates and thus are dietarily

nonessential.

2. Several reactions of IMP biosynthesis require folate derivatives and glutamine.

Consequently, antifolate drugs and glutamine analogs inhibit purine biosynthesis.

3. Oxidation and amination of IMP forms AMP and GMP, and subsequent

phosphoryl transfer from ATP forms ADP and GDP. Further phosphoryl transfer

from ATP to GDP forms GTP. ADP is converted to ATP by oxidative

phosphorylation. Reduction of NDPs forms dNDPs.

4. Hepatic purine nucleotide biosynthesis is stringently regulated by the pool size of

PRPP and by feedback inhibition of PRPP-glutamyl amidotransferase by AMP

and GMP.

5. Coordinated regulation of purine and pyrimidine nucleotide biosynthesis ensures

their presence in proportions appropriate for nucleic acid biosynthesis and other

metabolic needs.

6. Humans catabolize purines to uric acid (pKa 5.8), present as the relatively

insoluble acid at acidic pH or as its more soluble sodium urate salt at a pH near

neutrality. Urate crystals are diagnostic of gout. Other disorders of purine

catabolism include Lesch- Nyhan syndrome, von Gierke’s disease, and

hypouricemias.

7. Since pyrimidine catabolites are water-soluble, their overproduction does not

result in clinical abnormalities. Excretion of pyrimidine precursors can, however,

result from a deficiency of ornithine transcarbamoylase because excess

carbamoyl phosphate is available for pyrimidine biosynthesis.


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Lecture 9: Clinical cases and biochemical interpretations (1) Case 1: A 21-year-old college student presents to the clinic complaining of a sudden onset of chills and fever, muscle aches, headache, fatigue, sore throat, and painful nonproductive cough 3 days prior to fall final exams. Numerous friends of the patient in the dormitory reported similar symptoms and were given the diagnosis of influenza. He said that some of them were given a prescription for ribavirin. On examination, he appears ill with temperature 39.4°C (103°F). His skin is warm to the touch, but no rashes are appreciated. The patient has mild cervical lymph node enlargement but otherwise has a normal examination.

1. What is the most likely diagnosis? 2. What is the biochemical mechanism of action of ribavirin? 3. What is the genetic make up of this infectious organism?

ANSWERS TO CASE 1: RIBAVIRIN AND INFLUENZA Summary: A college student complains of the sudden onset of fever, chills, malaise, nonproductive cough, and numerous sick contacts in the fall season.

1. Likely diagnosis: Acute influenza infection 2. Biochemical mechanism of action of ribavirin: A nucleoside analogue with

activity against a variety of viral infections 3. Genetic makeup of organism: Ribonucleic acid (RNA) respiratory virus

CLINICAL CORRELATION This 21-year-old college student has the clinical clues suggestive of acute influenza. Typically, the illness occurs in the winter months with an acute onset of fever, myalgias (muscle aches), headache, cough, and sore throat. Usually, there are outbreaks with many individuals with the same symptoms. This patient is young and healthy, and antiviral therapy is not mandatory. The best way to prevent the infection is by influenza vaccination, usually given in October or November of each year. Because of the antigenic changes of the virus, a new vaccine must be given each year. Patients who are at especially high risk for severe complications or death should receive the vaccine each year. These include the elderly and people with asthma, chronic lung disease, human immunodeficiency virus (HIV) infection, diabetes, or chronic renal insufficiency. APPROACH TO THE USE OF RIBAVIRIN IN INFLUENZA Objectives

1. Know the structure of deoxyribonucleic acid (DNA) and RNA. 2. Know the differences between RNA and DNA. 3. Be familiar with the differences between human and viral/bacterial DNA and

RNA.


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COMPREHENSION QUESTIONS

1. Influenza virus is a class Vb virus, which means that it has a single (–)- stranded RNA for its genome. Which of the following best describes the immediate fate of this (–)-RNA when the virus enters the host cell?

A. It is used directly to encode viral proteins. B. It is used as a template to synthesize a (+)-strand viral messenger RNA

(mRNA). C. It is used as a template to synthesize viral DNA. D. It is converted to a provirus. E. It is integrated into the host cell genome.

2. If a double-stranded DNA molecule undergoes two rounds of replication in an

in vitro system that contains all of the necessary enzymes and nucleoside triphosphates that have been labeled with 32P, which of thefollowing best describes the distribution of radioactivity in the four esulting DNA molecules?

A. Exactly one of the molecules contains no radioactivity. B. Exactly one of the molecules contains radioactivity in only one strand. C. Two of the molecules contain radioactivity in both strands. D. Three of the molecules contain radioactivity in both strands. E. All four molecules contain radioactivity in only one strand.

3. A 48-year-old man has had a lengthy history of skin cancer. In the past

6 years he has had over 30 neoplasms removed from sun-exposed areas and has been diagnosed with xeroderma pigmentosum. Which of the following best describes the enzymatic defect in patients with xeroderma pigmentosum?

A. DNA polymerase α B. DNA polymerase γ C. DNA ligase D. Excision repair enzymes E. RNA polymerase III


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Lecture 10: Clinical cases and biochemical interpretations (2)

Case 2: A 32-year-old female presents to your clinic with concerns over a recently detected right breast lump. A mammogram performed revealed a right breast mass measuring 3 cm with numerous microcalcifications suggestive of breast cancer. During your discussion with the patient, she revealed that she had a sister who was diagnosed with breast cancer at the age of 39, a mother who passed away with ovarian cancer at age 40 years, and a maternal aunt who had both breast and colon cancer. Patient underwent an examination which revealed a fixed and nontender breast mass on right side measuring 3 cm with mild right axillary lymphadenopathy. No skin involvement is noted. A biopsy was performed and revealed intraductal carcinoma.

1. What cancer gene might be associated with this clinical scenario? 2. What is the likely mechanism of the cancer gene in this case?

ANSWERS TO CASE 2: ONCOGENES AND CANCER Summary: A 32-year-old female with strong family history of breast, colon, and ovarian cancer, who now presents with a fixed breast lesion that is biopsyproven carcinoma.

1. Most likely cancer gene: Breast cancer (BRCA) gene 2. Likely mechanism: Inhibition of tumor-suppressor gene

CLINICAL CORRELATION This young woman has developed breast cancer at age 32 years. Moreover, she has two first-degree relatives with breast and/or ovarian cancer prior to menopause. This makes BRCA gene mutation likely. The BRCA1 gene resides on chromosome 17. This gene encodes a protein which most likely is important in deoxyribonucleic acid (DNA) repair. Thus, a mutation of the BRCA1 gene likely leads to abnormal cells propagating unchecked. A woman with a BRCA1 mutation has a 70 percent lifetime risk of developing breast cancer, and a 30 to 40 percent risk of ovarian cancer. The vast majority of breast cancer is not genetically based, but occurs sporadically. However, familial-based breast cancers are most common because of BRCA1 mutation. BRCA2 is another mutation that is more commonly associated with male breast cancer. Other genetic mechanisms of cancer include oncogenes, which are abnormal genes that cause cancer usually by mutations. Protooncogenes are normal genes that are present in normal cells and involved in normal growth and development, but if mutations occur, they may become oncogenes. APPROACH TO ONCOGENES Objectives 1. Know the definitions of oncogenes and protooncogenes. 2. Understand the role of promoter and repressor functions of DNA synthesis. 3. Know the normal DNA replication. 4. Be familiar with DNA mutations (point mutations, insertions, deletions). 5. Know the process of DNA repair. 6. Understand the recombination and transposition of genes.


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COMPREHENSION QUESTIONS

1. Hereditary retinoblastoma is a genetic disease that is inherited as an autosomal dominant trait. Patients with hereditary retinoblastoma develop tumors of the retina early in life, usually in both eyes. The affected gene (RB1) was the first tumor suppressor gene to be identified. Which of the following best describes the function of the protein encoded by the RB1 gene?

A. It binds transcription factors required for expression of DNA replication enzymes.

B. It allosterically inhibits DNA polymerase. C. It binds to the promoter region of DNA and prevents transcription. D. It phosphorylates signal-transduction proteins.

2. Mutations in the tumor suppressor gene BRCA1 are transmitted in an

autosomal dominant fashion. When a cell is transformed to a tumor cell in individuals who have inherited one mutant allele of this tumor suppressor gene, which of the following most likely occurs?

A. A transcription factor is over expressed. B. Deletion or mutation of the normal gene on the other chromosome. C. Chromosomal translocation. D. Gene duplication of the mutant gene.

3. Women who inherit one mutant BRCA1 gene have a 60 percent chance of

developing breast cancer by the age of 50. The protein produced by the BRCA1 gene has been found to be involved in the repair of DNA double-strand breaks. Which of the following processes is most likely to be adversely affected by a deficiency in the BRCA1 protein?

A. Removal of thymine dimers B. Removal of RNA primers C. Removal of carcinogen adducts D. Homologous recombination E. Correction of mismatch errors

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