MB 207 – Molecular Cell Biology
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Transcript of MB 207 – Molecular Cell Biology
MB 207 – Molecular Cell Biology
DNA Damage and Repair
DNA recombination
DNA Damage and Repair
• Maintaining genetic stability is very important - accurate mechanism for replicating DNA. - mechanism for repairing DNA alterations that arise both
spontaneously and from exposure to DNA-damaging environmental agents.
• Nearly all DNA damage is harmful but occasionally beneficial because mutations provide genetic variability.
How important is DNA repair?– DNA is the only biomolecule that is specifically repaired. All others
are replaced. – DNA damage is repaired shortly after it occurs and hence it does not
affect future generations– >100 genes participate in various aspects of DNA repair, even in
organisms with very small genomes. – Cancer is caused by mutations as well as many other diseases.
DNA damage
Spontaneous Mutagens
Depurination Deamination Chemicals Radiation
Spontaneous mutations
• Hydrolysis reactions caused by random interactions between DNA and the molecules around it.
• Two types of spontaneous mutations: Depurination Deamination
• Depurination
the loss of a purine base by spontaneous hydrolysis of glycosidic bond that links it to deoxyribose.
this glycosidic bond is labile under physiological conditions. Therefore, susceptible to hydrolysis that DNA loss thousands of purine bases in the human cell everyday.
• Deamination
Primary amino groups of nucleic acid bases are unstable. They can be converted to keto groups in the hydrolysis reactions and become deaminated.
Involve cytosine, adenine and guanine, changes the base pairing properties of the affected base.
Cytosine is more susceptible to deamination, giving rise to uracil. Others: Adenine to Hypoxanthine, Guanine to Xanthine, and 5-methyl cytosine to Thymine.
Usually caused by random collision of a water molecule with the bond that links the amino group of the base to the pyrimidine or purine ring.
Rate is about 100 deaminations per day. If not repaired, the error base sequence may be propagated when
the strand serves as a template in the next round of replication.
Depurination & Deamination
Deamination of DNA nucleotides
A. Deamination of cytosine
produces uracil
Results in the substitution of one base for another when the DNA is replicated
Missing purine
B. Depurination
If uncorrected, can lead to either the substitution or the loss of a nucleotide pair.
Mutagens (mutation-causing agents)
• Two major categories Chemicals Radiations
• Chemicals - alter DNA structure by a variety of mechanisms.
Base analogs
- resemble nitrogenous bases in structure and are incorporated into DNA.
Base modifying agents
- reacts chemically eith DNA bases to alter their structures.
Intercalating agents
- insert themselves between adjacent bases of the double helix.
• Radiations
Sunlight (ultraviolet radiation)
- alters DNA by triggering pyrimidine dimer formation (formation of covalent bonds between adjacent pyrimidine bases).
- blocked replication and transcription.
X-rays and related form of radiation emitted by radioactive substances
- ionizing radiation because it removes electrons from biological molecules.
- generating highly reactive intermediates that cause various types of DNA damage.
DNA damages
• Distortion of double helix structure– Photodamage
• UV light absorbed by the nucleic acid bases can induce bond formation between adjacent pyrimidines (C or T) within one strand.
• The two adjacent pyrimidines are pulled closer to each other than in normal DNA
• Strand breaks– Single-strand and double-strand
breaks are produced at low frequency during normal DNA metabolism by topoisomerases, nucleases and repair processes as well as by ionizing radiation.
The thymine dimer
This type of damage is introduced into DNA in cells that are exposed to ultraviolet irradiation
Types of DNA damages
• Spontaneous oxidative damage (red arrows)
• hydrolytic attack (blue arrows)
• Uncontrolled methylation (green arrows))
DNA Repair Mechanism
DIRECT REVERSAL
DOUBLE STRAND BREAKS
TRANSLESION SYNTHESIS
SINGLE STRAND DAMAGE
BASE EXCISION
REPAIR
MISMATCH REPAIR
NUCLEOTIDE EXCISION
REPAIR
NON-HOMOLOGOUS
END JOINING
HOMOLOGOUS RECOMBINATION
Repairing Damaged BasesDamaged or inappropriate bases can be repaired by several mechanisms:
1. Direct chemical reversal of the damage
2. Excision Repair, in which the damaged base or bases are removed and then replaced with the correct ones in a localized burst of DNA synthesis. There are three modes of excision repair, each of which employs specialized sets of enzymes. Base Excision Repair (BER) Nucleotide Excision Repair (NER) Mismatch Repair (MMR)
3. Double strand breaks
Non-homologous end joining
Homologous recombination
4. Translesion synthesis- DNA damage tolerance process that allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites
1. Direct Reversal of Base Damage
The most frequent cause of point mutations in humans is the spontaneous addition of a methyl group (CH3-) (an example of alkylation) to Cs followed by deamination to a T. Fortunately, most of these changes are repaired by enzymes, called glycosylases, that remove the mismatched T restoring the correct C. This is done without the need to break the DNA backbone (in contrast to the mechanisms of excision repair described below).
Some of the drugs used in cancer chemotherapy ("chemo") also damage DNA by alkylation. Some of the methyl groups can be removed by a protein encoded by our MGMT gene. However, the protein can only do it once, so the removal of each methyl group requires another molecule of protein.
This illustrates a problem with direct reversal mechanisms of DNA repair: they are quite wasteful.
2. Excision Repair
A. Base excision repair (BER), which repairs damage to a single nucleotide caused by oxidation, alkylation, hydrolysis, or deamination. The base is removed with glycosylase and ultimately replaced by repair synthesis with DNA ligase.
B. Nucleotide excision repair (NER), which repairs damage affecting longer strands of 2–30 bases. This process recognizes bulky, helix-distorting changes such as thymine dimers as well as single-strand breaks (repaired with enzymes such UvrABC endonuclease). A specialized form of NER known as Transcription-Coupled Repair (TCR) deploys high-priority NER repair enzymes to genes that are being actively transcribed.
C. Mismatch repair (MMR), which corrects errors of DNA replication and recombination that result in mispaired (but normal, that is non- damaged) nucleotides following DNA replication
DNA repair mechanisms
1) Base excision repair (BER)– Removal of the incorrect base by an
appropriate DNA glycosylase to create a deoxyribose sugar lacking it’s base (AP site - apurinic / apyrimidinic)
– Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, thus creating a 3'-OH terminus adjacent to the AP site, removal of sugar phosphate.
– Extension of the 3'-OH terminus by a DNA polymerase, DNA ligase seals nick.
– e.g. removal of uracil from DNA
Oxidation of guanine
DNA repair mechanisms
2) Nucleotide excision repair (NER)– Removes a whole oligonucleotide
that contain the damage. – Steps:
• Multienzyme complex recognizes damaged regions based on their abnormal structure as well as on their abnormal chemistry (eg. pyrimidine dimer)
• Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides
• An associated DNA helicase removes the entire damaged strand, in-between the nicks.
• Bacteria multienzyme complex leaves a 12nt gap; doubles the size in human DNA
• Filling in of the resulting gap by a DNA polymerase
• Ligation by DNA ligase.
(bulky lesion)
Repairing Strand Breaks
Ionizing radiation and certain chemicals can produce both single-strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA backbone.
A. Single-Strand Breaks (SSBs)
Breaks in a single strand of the DNA molecule are repaired using the same enzyme systems that are used in Base-Excision Repair (BER).
B. Double-Strand Breaks (DSBs)
There are two mechanisms by which the cell attempts to repair a complete break in a DNA molecule:
i. Direct joining of the broken ends.
-This requires proteins that recognize and bind to the exposed ends and bring them together for ligasing. They would prefer to see some complementary nucleotides but can proceed without them so this type of joining is also called Nonhomologous End-Joining (NHEJ).
-Errors in direct joining may be a cause of the various translocations that are associated with cancers.
(Translocation: Type of mutation in which a portion of 1 chromosome is broken off and attached to another)
ii. Homologous Recombination. Here the broken ends are repaired using the information on the intact
-sister chromatid (available in G2 after chromosome duplication), or on the
-homologous chromosome (in G1; that is, before each chromosome has been duplicated). This requires searching around in the nucleus for the homolog — a task sufficiently uncertain that G1 cells usually prefer to mend their DSBs by NHEJ. or on the
-same chromosome if there are duplicate copies of the gene on the chromosome oriented in opposite directions (head-to-head or back-to-back).
-Two of the proteins used in homologous recombination are encoded by the genes BRCA1 and BRCA2. Inherited mutations in these genes predispose women to breast and ovarian cancers.
DNA repair mechanisms
Two different types of end-joining for repairing double-strand breaks
1. Nonhomologous end-joining– permits joining of double-strand breaks even if there is no sequence
similarity between them – Broken ends are rejoined by DNA ligation with the loss of one or
more nucleotides at the joining site– Alters the original DNA sequence either by deletions or short
insertions.
2. Homologous end-joining– More difficult to accomplish but is more precise– cells are diploid – contain 2 copies of each double helix– Recombination mechanisms used to transfer nucleotide sequence
information from the homologous intact DNA double helix to the site of the double-strand break
• Both system involve a lot of different proteins and the processes are much more complicated
DNA end-joining for repairing ds breaks
Accidental break (ionizing radiation, Accidental break (ionizing radiation, oxidizing agents, replication errors)oxidizing agents, replication errors)
Loss of nucleotides due to Loss of nucleotides due to degradation from endsdegradation from ends
Region with altered segment Region with altered segment due to missing nucleotidesdue to missing nucleotides
Nonhomologous end-joining- Common in mammalian cells
Homologous end-joining
Copying process involving Copying process involving homologous recombinationhomologous recombination
Complete sequence restored by copyingComplete sequence restored by copying from second chromosomefrom second chromosome((replication process uses the undamaged chromosome as the template for transferring genetic information to the broken chromosome, repairing it with no change in the DNA sequences))
DNA ligation
Summary of DNA repair systems
Type Damage Enzyme
Mismatch repair Replication errors MutS, MutL, and MutH in E. coli
MSH, MLH and PMS in humans
Photoreaction Pyrimidine dimers DNA photolyase
Base excision repair
Damaged base DNA glycosylase
Nucleotide excision repair
Pyrimidine dimerBulky adduct on base
UvrA, UvrB, UvrC and UvrD in E. coli
XPC, XPA, XPD, ERCI-XPF and XPG in humans
Double strand break repair
Double strand breaks RecA and RecBCD in E.coli
Translesion DNA synthesis
Pyrimidine dimer or apurinic site
Y-family DNA polymerase, such as UmuC in E. coli
DNA Recombination
• A process that a DNA segment moves from one DNA molecule to another DNA molecule– DNA molecules recombine by breaking and rejoining– Phosphodiester bonds are broken and rejoined.
• Importance of DNA recombination:– the process of introducing genetic variation: Genetic variation is
crucial to allow organisms to evolve in response to a changing environment. E.g., genetic recombination results in the exchange of genes between paired homologous chromosomes during meiosis.
– an important mechanism for repairing damaged DNA.– involved in rearrangements of specific DNA sequences that alter the
expression and function of some genes during development and differentiation.
• Two broad classes are commonly recognized - general recombination & site-specific recombination.
A heteroduplex joint General recombination in meiosis
General Recombination• Allow large section of the DNA double
helix to move from one chromosome to another
• Responsible for the crossing-over of chromosomes during meiosis
• Chromosome must synapse (pair) in order for chiasmata to form where crossing-over occurs– DNA synapsis: base pairing between
complementary strands from 2 DNA molecules
– Chiasmata: regions where paired homologous chromosomes exchange genetic material during meiosis, a cross-shaped structure
• Only occurs between homologous DNA molecules
General Recombination
• Two homologous DNA molecules line up.• Nicks (single or double??) are introduced.• Each nicked strand then invades the other DNA molecule by
complementary base pairing.
• The cut strands cross and join homologous strands, forming the Holliday structure (or Holliday junction) (R. Holliday (1964).
• Once a Holliday junction is formed, it can be resolved 2 ways by nicking and rejoining of the crossed strands to yield 2 different heteroduplexes: – recombinant heteroduplexes: resulting DNA molecules are a
combination of both parental DNA molecules.– non-recombinant heteroduplexes: resulting DNA molecules
contain only DNA from one parent molecule with a small portion of heteroduplex.
Non-recombinant Recombinant
Paternal chromosome A
Maternal chromosome B
Holliday junction cleavage
‘splice’ or crossover products reassortment or flanking genes
‘patch’ or non- crossover products no reassortment
DNA clearage
DSB repair model for homologous recombination. The figure shows the step leading to generation of recombination intermediate with 2 Holliday junctions.
General Recombination: example
• Various enzymes (homologues) are involved in the recombination process:
– Rec A: catalyze the exchange of strands between homologous DNAs that causes heteroduplexes to form
– RecB, C, & D: complex of three proteins1. acts as a helicase and transiently unwinds the double-stranded DNA2. When it encounters the specific nucleotide sequence GCTGGTGG (the
chi site), the enzyme acts as a nuclease to introduce a single-stranded nick
3. Continue to unwind the double helix, forming a displaced single strand to which RecA can bind to initiate strand exchange.
– Ruv A, B: catalyze the movement of the crossed-strand site in Holliday junctions
– RuvC: resolves the Holliday junction by cleaving the crossed strands, which are then joined by ligase
Site-Specific Recombination
• Occurs between sequences with a limited stretch of similarity; involves specific sites
• Mediated by proteins that recognize the specific DNA target sequences rather than by complementary base pairing
• Transposons/transposable elements/ “Jumping genes“: mobile genetic elements that can move throughout the genome
• Two distinct mechanisms:1. Transpositional site-specific recombination: insertion of mobile
genetic elements into any DNA sequence, no formation of heteroduplex
2. Conservative site-specific recombination: site specific recombination that requires a short DNA sequence that is the same on both donor and recipient, involve formation of heteroduplex
Transposon (cont’ next)• Transposase / Integrase: act on the specific sequence at the end of
transposon and disconnecting it from the flanking sequence and then inserting it to a new target site
Cut and Paste transposition (DNA-only transposons )
Steps of cut and paste transposition:
1. Binding of transposase subunits to the terminal inverted repeats
2. Transpososome formation (synaptic complex)
3. Excision of the transposon (contrast to replicative mechanism)
4. DNA strand transfer
5. Gap repair – DNA polymerase
Replicative transposition (DNA-only transposons)
nick
Steps of Replicative transposition mechanism:
1. Binding of transposase subunits to the terminal inverted repeats
2. Transpososome formation
3. Cleavage to generate 3’ OH groups. Transposon DNA is not excised from host
4. DNA strand transfer
5. Replication (cointegrate with 2 copies of transposon)
Retrovirus-like transposition
1. LTR on the two ends of the element
2. Transcription to generate RNA copiy
3. RNA template to synthesize DNA using reverse transcriptase
4. cDNA is recognized by integrase
5. Gap repair
Non-retroviral transposition-poly-A retrotransposons move by a ‘Reverse Splicing’ mechanism
called target site primed reverse transcription
• A significant fraction of vertebrate chromosomes is made up of repeated DNA sequences
• In human chromosomes, these repeats are mostly mutated/truncated versions of a retrotransposon called L1 element (LINE= lone interspersed nuclear element)
• L1 element are mostly immobile• Translocation result in human disease eg. Hemophilia – L1 insertion into a gene for blood clotting
factor VIII.• Mechanism: require a complex of endonuclease and a
reverse transcriptase
Non-retroviral transposition
Generates a ssDNA element directly linked to target DNA
Processing of ssDNA to produce dsDNA of L1
Conservative site-specific recombination- Breaking and joining occur at two special sites, one on each participating DNA molecules- enzymes involve can break and rejoin two DNA helix, often reversible, ie. DNA integration, DNA excision or DNA inversion can occur- eg. Bacteriophage lambda/ bacterial viruses – mobile DNA element, moving in and out of host chromosomes
eg. Salmonella typhimuriumInversion of DNA segment changes the type of flagellum produced
eg. Bacteriophage
Conservative site-specific recombinationInsertion of a circular bacteriophage lambda DNA chromosome into bacteria chromosome:
1. Integrase binds to specific ‘attachment site’ on each chromosome
2. Cuts and switch the partner strands
3. Re-seals forming heteroduplex joint (7nt bp long)
4. Phosphodiester bond breakage release energy used for strand joining
5. Intergrase dissociates.
How a conservative site-specific recombination enzyme is used to turn on a specific gene in a group of cells in a transgenic animal:
(used in mice or Drosophila to study the effect of expressing a gene of interest in the animal, using Cre recombination enzyme & loxP recognition sites)
Major types of transposable elements
Type Structural features Mechanism of movement
DNA-mediated transposition
Bacterial replicative transposons Terminal inverted repeats that flank antibiotic resistance and transposase genes
Copying of element DNA accompanying each round of insertion into a new target site
Bacterial cut and paste transposons
Terminal inverted repeats that flank antibiotic resistance and transposase genes
Excision of DNA from old target site and insertion into new site
Eukaryotic transposons Inverted repeats that flank coding region with introns
Excision of DNA from old target site and insertion into new site
RNA-mediated transposition
Viral-like retrotransposons ~250 to 600bp direct terminal repeats (LTRs) flanking genes for reverse, transcriptase, integrase and retroviral-like Gag protein
Transcription into RNA from promoter in left LTR by RNA polymerase II followed by reverse transcription and insertion at target site
Poly-A retrotransposons 3’ A-T rich sequence and 5’ UTR flank genes encoding an RNA-binding protein and reverse transcriptase
Transcription into RNA from internal promoter; target primed reverse transcription initiated by endonuclease cleavage