Lecture 8 - DNA Replication PDF

Lecture 8 – DNA replication Part 1 – The most beautiful experiment How is DNA organised? E.g. Escherichia coli E.coli Genome Compaction Genome size: 4639 kb, circular double-stranded DNA. Base pair spacing: 0.34 nm. Total DNA length: 1.6 mm. Cell size: 1–2 µm. Compac...

Lecture 8 – DNA replication Part 1 – The most beautiful experiment How is DNA organised? E.g. Escherichia coli E.coli Genome Compaction Genome size: 4639 kb, circular double-stranded DNA. Base pair spacing: 0.34 nm. Total DNA length: 1.6 mm. Cell size: 1–2 µm. Compaction: Supercoiling: DNA twisted to reduce size. Nucleoid-associated proteins help compact DNA. DNA is tightly packed to fit inside the small bacterial cell. Our DNA Human genome: 6.4 Gbp of DNA in each somatic cell. Total DNA length: ~2.2 m per cell. Nucleus size: ~6 µm diameter. DNA is compacted through nucleosomes and chromatin fibres. Body DNA length: Enough to stretch to the Sun and back ~730 times. Our DNA is packed more tightly than E. coli’s Eukaryotic DNA organised DNA replication DNA must be decompacted and then copied faithfully during cell division The error rate is very low Watson-Crick model – mechanism of replication 1953 – Watson – Crick noticed that the specific base pairing suggests possibility of copying mechanisms for the genetic material Each DNA strand can act as a template to build a complimentary copy, and the daughter molecule would have the same sequence as the parental molecules. Watson-Crick model – two predictions 1. DNA strands are held together by ‘Watson-Crick’ base-pairing, consistent with Chargaff’s rules A pairs with T and T with A: 2H bonds G pairs with C and C with G: 3H bonds 2. Each strand is therefore complementary to the other, so each can act as a template for DNA replication: DNA replication should therefore be semi-conservative Both of these predictions were tested by others between 1957 and 1961. Testing DNA replication: other models 1. Semi-conservative: Each daughter DNA molecule contains one parental strand and one newly replicated strand. WATSON-CRICK model 2. Conservative: The parent helix is conserved, whilst the daughter helix is completely new. 3. Dispersive: parent helix is broken into fragments, dispersed, copied and then assembled into two new helices. Meselson and Stahl – 1958: testing models of DNA replication They described caesium chloride (CsCl) equilibrium density gradient centrifugation: a technique that separates molecules based on their densities. Macromolecules are suspended in a CsCl solution with a density approximately equal to that of the macromolecules. The solution is then spun, forming a CsCl density gradient. Establishing the conditions Two types of DNA (14N-DNA and 15N-DNA) are mixed with caesium chloride (CsCl) which creates a density gradient during centrifugation. DNA is separated using high-speed centrifugation at 140,000 g for 20 hours, with heavy DNA settling lower and light DNA higher in the gradient. The DNA bands are illuminated with UV light after centrifugation, allowing them to appear distinct against the background, and a photograph is taken to showcase the different types of DNA. Therefore, comparing the densities of DNA from different conditions, the experiment confirmed that DNA replication is semi-conservation, with each new DNA molecule consisting of one original strand and one newly synthesized strand. The prediction The reality Only three forms of DNA were seen: H, hybrid and L: after one generation, all the DNA was hybrid. For later generations there was a gradual loss of hybrid 14N/15N DNA and replication with light DNA. Therefore, DNA replication is semi-conservative. Part 2 – The discovery of DNA polymerases 1957 – Arthur Kornberg and the discovery of DNA polymerase 1941: Beadle and Tatum: ‘one gene-one enzyme Therefore, there was an expectation that there would be enzyme capable of replicating DNA: a DNA polymerase Kornberg took Escherichia coli protein extracts, and worked out the in vitro conditions that were required for this extract to make new DNA: 1. A template DNA to copy 2. deoxyucleotide triphosphates (dATP, dTTP, dCTP and dGTP) 3. the co-factor Mg2+ 4. an energy source, ATP 5. … and a small piece of DNA (a PRIMER) with a free 3’ OH Conclusion: new strands are extended in the 5’--> 3’ direction by a polymerase. Pol I have other activities as well 5’--> 3’ DNA polymerizing activity 3’--> 5’ exonuclease activity 5’--> 3’ exonuclease activity This suggests that Pol I has editing/proof reading functions and can correct mistakes. But Pol I is Not the enzyme that is used for replication of E. coli DNA Pol III is esstinal for DNA replication in E. coli It was discovered by Thomas Kornberg (son of Arthur Kornberg) ‘ Conclusions, so far... New DNA is made in the 5’ → 3’ direction and is mediated by polymerases, which can also edit mistakes. This doesn’t really test the Watson-Crick model … Nucleotide incorporation to test DNA strand polarity (1) If the sugar-proximal phosphate is radioactive then a radioactive phosphate will be inserted 5’ of the new nucleotide residue incorporated. Testing DNA strand polarity (2) Testing DNA strand polarity (3) 1. The proportion of radiolabelled Ts in his DNA sample was 0.061 2. Labelling with dATP allowed him to measure GpA. Editing by DNA polymerase Conclusion, so far The DNA strands are anti-parallel Watson-Crick base pairing is correct DNA replication is semi-conservative New DNA is made in the 5’ → 3’ direction and is mediated by polymerases that can edit errors, but which require priming …… and this causes a problem The problems 1. Replication requires priming 2. The primers are extended, growing in the 5’ --> 3’ direction 3. Because new DNA is built 5’ --> 3’ this strand needs new priming and extension (lagging strand) 4. Whereas this strand supports continual synthesis (leading strand)