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MCD Nucleic Acids Alexandra Burke-Smith
1
1. Nucleic Acids and Chromosomes Dr Birgit Leitinger ([email protected])
DNA and RNA bases - purines and pyrimidines, the four bases of DNA (A, T, G, C) and of RNA (A, U, G, C)
Sugars in DNA and RNA - deoxyribose and ribose
Nucleotides and nucleosides
DNA: polymer of deoxyribonucleotide units
Primary sequence of DNA specifies genetic information
DNA double helix - antiparallel complementary strands
Melting and annealing of complementary strands
Watson-Crick base pairing: G-C and A-T
Genomes of E.coli (a single circular DNA molecule) and Homo Sapiens (46 chromosomes contain linear DNA)
The human karyotype
Packaging of eukaryotic DNA - chromatin and chromosomes
1. Outline the Chemical composition of DNA
Nucleic Acids
DNA and RNA are nucleic acids; linear polymer chains consisting of nucleotides joined by phosphodiester
bonds
DNA is a linear polymer of deoxyribonucleotide units
A nucleotide consists of a nitrogenous base, a sugar and one or more phosphate groups
A nucleoside is composed of a nitrogenous base and a sugar (NO PHOSPHATE)
DNA and RNA each contain 4 different types of nucleotides that are arranged in different sequences.
Sugars- deoxyribose or ribose
The sugar in DNA is deoxyribose, lacking an oxygen atom that is present in ribose, the parent compound. Ribose is the sugar in RNA.
Primes () are used in numbering the carbon atoms in the ribose: 1 to 5. The 1 C is linked to the base; the 5 C is linked to the phosphate. The 3 C has an OH group which is important in the structure of DNA.
DNA and RNA nitrogenous bases
There are 5 different bases; but each type of nucleic acid (DNA or RNA) contains only 4 bases:
Purines (big): Adenine (A) and Guanine (G)
Consists of TWO aromatic rings including carbon and nitrogen
Pyrimidines (Small): Cytosine (C) and Thymine (T)/ Uracil (U)- in RNA
Consists of ONE aromatic ring including carbon and nitrogen
The nitrogenous bases are then linked to the pentose sugar (ribose or doxyribose) by a beta-Glycosidic
linkage
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MCD Nucleic Acids Alexandra Burke-Smith
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Nucleosides vs Nucleotides
Base + (deoxy)ribose = nucleoside
Base Nucleoside
Adenine (deoxy)adenosine
Guanine (deoxy)guanosine
Cytosine (deoxy)cytidine
Uracil (deoxy)uridine
Thymine (deoxy)thymidine
Note: a nucleoside can also involve a ribose sugar instead of a deoxyribose, e.g. adenosine
Base + ribose + phosphate = nucleotide
E.g. adenosine monophosphate (AMP), deoxycytidine triphosphate (dCTP)
2. Describe the Structure of DNA
A single DNA strand is a long chain of deoxyribonucleotide units linked by phosphodiester links.
The 3 C-OH group on a sugar of one nucleotide is linked to phosphate group, which in turn is joined to 5-OH of an adjacent sugar.
On each deoxyribose there is a base.
The chain has two ends, the 5 end and the 3 end. It is not symmetrical.
The primary sequence is the linear sequence of the bases. By convention, the nucleotide sequence is specified in the 5 to 3 direction.
The bases carry genetic information, the sugar and phosphates perform a structural role.
The secondary structure of DNA is a right-handed double helix. The two chains in the helix run in opposite directions; antiparallel
There are 10 base pairs per helical turn
The deoxyribose and phosphate groups run along the outside of the helix, with the negative charges outside.
The bases point inwards and the flat planes are perpendicular to the helix.
The two chains are held together by hydrogen bonds between the bases.
The two strands are complementary in their sequence due to the specificity of base-pairing. Adenine always pairs with Thymine (or Uracil in RNA); Guanine always pairs with Cytosine.
A and T form 2 Hydrogen bonds, which is less stable than C and G, which forms 3 hydrogen bonds
The DNA strands can be separated by Melting (Heat or low salt) and re-annealing (Cool or high salt)
RNA can assume a variety of shapes.
3. Explain How cells package DNA
Genome The entire DNA coding for an organism constitutes its genome.
E.coli has 4.6 x 106 base pairs in a single circular double-stranded molecule. The length of E.coli DNA is 1.4
mm.
The human genome is approxiamately 3 x 109 base pairs of DNA divided into chromosomes that each
contain a single, linear double-helical DNA molecule of ~ 200 x 106 base pairs. Chromosomes are visible only just before cells divide but not in non-dividing cells. Mitotic chromosomes: highly condensed form of
chromosomes. Interphase chromosomes: extended form
MCD Nucleic Acids Alexandra Burke-Smith
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The karyotype A karyotype is an organised profile of someones chromosomes.
A diploid human cell has 46 chromosomes o 22 pairs of normal chromosomes (autosomes) and o 2 sex chromosomes (X and Y)
Abnormal Karyotypes in breast tumour: 48 chromosomes instead of 46, multiple translocations, eg two pieces of
chromosome 8 (green) plus piece of chromosome 17 (purple).
Packaging of eukaryotic DNA
tightly packaged, forming a complex with proteins. This complex is called chromatin. During interphase,
when cells are not dividing, chromatin is more extended. Further condensation during mitosis produces
chromosomes.
The lowest level of packaging is the nucleosome, which consists of DNA wrapped around histone proteins.
Nucleosome includes ~ 200 bp DNA (linker DNA plus core DNA)
Structure of the nucleosome
The nucleosome causes an approximately 7-fold condensation of DNA. (200 bp ~68nm; wrapping around
histone octamer ~10 nm).
The chain of nucleosomes is further packed to generate a more compact structure: the 30 nm fiber, giving
~40-fold condensation.
In the interphase nucleus the DNA is further condensed about 100-1000 fold.
Chromosomes
MCD Nucleic Acids Alexandra Burke-Smith
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2. DNA Replication, the cell cycle and mitosis Dr Birgit Leitinger ([email protected])
DNA replication is semi-conservative
DNA synthesis is mediated by DNA polymerases
DNA polymerases require a template and a primer
DNA synthesis occurs in the 5 to 3 direction
Nucleoside analogs lacking a 3-OH terminate the chain
The replication fork, leading and lagging strands
Proof reading mechanism
Replication of E.coli and mammalian chromosomes
Phases of the life cycle of a eukaryotic cell
Stages of mitosis
1. Explain how cells duplicate DNA
DNA replication
Before each cell division, a faithful copy of the DNA needs to be made to ensure
genetic continuity in the daughter cell
DNA replication is semi-conservative. Each daughter cell inherits one old and
one new strand.
The two strands are complementary to each other so each strand serves as a
template for the synthesis of the other strand. This generates two identical copies.
The DNA helix is very stable and has to be unwound before replication can occur. This is done by a DNA
helicase, an enzyme that uses ATP as source of energy to break hydrogen bonds between base pairs.
New DNA is synthesised by enzymes called DNA polymerases. DNA polymerases add nucleotides to the 3
end of a growing chain. DNA polymerases require a template strand, an olinucleotide primer (sequence of
polymerized nucleotides made by a different enzyme) , and a supply of of deoxynucleotide triphosphates
(dNTPs)
The Enzyme reaction
DNA polymerases add dNTPs to the 3 end of a DNA molecule.
DNA (and RNA) synthesis occurs in 5 to 3 direction.
Energy is released by hydrolysis of the triphosphate. This drives the reaction.
A free 3 hydroxyl group is required.
Some nucleoside analogs are used as drugs- they act as chain terminators e.g. Acyclovir- herpes, and
dideoxycytosine (ddC)- Drug for HIV (zalcitabine). Chain terminators do not have a free 3 hydroxyl group,
therefore polymerase cannot add anymore free dNTPs
The Replication Fork
Replication begins at discrete points on the DNA molecule called origin of replication.
The site of DNA synthesis is called a replication fork: the fork moves along during the process.
The templates for the two new daughter strands have opposite orientations: 3 to 5 and 5 to 3
The replication fork is asymmetric. Both strands are synthesised in a 5-3 direction. The leading strand is
synthesised continuously (i.e. the top one in the diagram above), whereas the lagging strand is synthesised
in short pieces termed Okazaki fragments
(bottom strand)
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MCD Nucleic Acids Alexandra Burke-Smith
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RNA Primers
RNA primes the synthesis of new DNA
A specialised RNA polymerase called DNA primase synthesises a short RNA fragment (~ 5 nucleotides). The RNA primer is only transient and removed at a later stage of replication.
For the synthesis of the leading strand, an RNA primer is needed only to start replication at a replication origin.
Synthesis of the Lagging Strand
DNA primase synthesises multiple short RNA fragment primers
DNA polymerase adds to RNA primer, starting the Okazaki fragment,
