1. DNA replication Learning objectives
Understand the basic rules governing DNA replication
Introduce proteins that are typically involved in generalised replication
Reference: Any of the recommended texts
Optional
Nature (2003) vol 421,pp

2. `It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’
Watson & Crick
Nature (1953)
Original drawing by Francis Crick

3. Four requirements for DNA to be genetic material
Must carry information
Cracking the genetic code
Must replicate
DNA replication
Must allow for information to change
Mutation
Must govern the expression of the phenotype
Gene function

4. DNA stores information in the sequence of its bases
Much of DNA’s sequence-specific information is accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it.
Some genetic information is accessible even in intact, double-stranded DNA molecules
Some proteins recognize the base sequence of DNA without unwinding it.
One example is a restriction enzyme.

5. DNA Replication
Process of duplication of the entire genome prior to cell division
Biological significance
extreme accuracy of DNA replication is necessary in order to preserve the integrity of the genome in successive generations
In eukaryotes , replication only occurs during the S phase of the cell cycle. The slower replication rate in eukaryotes results in a higher fidelity or accuracy of replication in eukaryotes

6. Basic rules of replication
Semi-conservative
Starts at the ‘origin’
Can be uni or bidirectional
Semi-discontinuous
Involves DNA templating
RNA primers required

7. A) DNA replication – three possible models
Semiconservative replication – Watson and Crick model
Conservative replication: The parental double helix remains intact; both strands of the daughter double helix are newly synthesized
Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material.

8. Meselson-Stahl experiments confirm semiconservative replication
Semi-conservative replication An ingenious experiment by Meselson and Stahl showed that one strand of each DNA strand is passed on unchanged to each of the daughter cells. This 'conserved' strand acts as a template for the synthesis of a new, complementary strand by the enzyme DNA polymerase
Figure 6.16

9. Figure 6.15

10. Meselson-Stahl experiments confirm semiconservative replication
Figure 6.16

11. Each strand acts as template
Double helix unwinds
Each strand acts as template
Complementary base pairing
Two daughter helices produced after replication
Figure 6.14

12. Starts at origin
Initiator proteins identify specific base sequences on DNA
Prokaryotes – single ori site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating sequences)
Prokaryotes
Eukaryotes

13. Uni or bidirectional

14. Bidirectional replication
Replication forks move in opposite directions
In linear chromosomes, telomeres ensure the maintenance and accurate replication of chromosome ends
In circular chromosomes, such as E. coli, there is only one origin of replication.
In circular chromosomes, unwinding and replication causes supercoiling, which may impede replication
Topoisomerase – enzyme that relaxes supercoils by nicking strands

15. The bidirectional replication of a circular chromosome
Figure 6.18 amb

16. Figure 6.18 c - f

17. Semi-discontinuous replication
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5’– 3’
Lagging strand synthesis in fragments in 5’-3’

18. Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction

19. DNA templating Nucleotide recognition Enzyme catalysed polymerisation
Complementary base pair copied
Substrate used is dNTP

20. RNA primers required

21. Core proteins at the replication fork
Topoisomerases
Helicases
Single strand
binding proteins
Primase
DNA polymerase
Tethering protein
DNA ligase
- Prevents torsion by DNA breaks
- separates 2 strands
- prevent reannealing
of single strands
- RNA primer synthesis
- synthesis of new strand
- stabilises polymerase
- seals nick via phosphodiester linkage

22. The mechanism of DNA replication
Arthur Kornberg, a Nobel prize winner and other biochemists deduced steps of replication
Initiation
Proteins bind to DNA and open up double helix
Prepare DNA for complementary base pairing
Elongation
Proteins connect the correct sequences of nucleotides into a continuous new strand of DNA
Termination
Proteins release the replication complex

23. Figure 6.17

24. Figure 6.17

25. Core proteins at the replication fork
Nature (2003) vol 421,pp
Figure in ‘Big’ Alberts too

26. Topoisomerase I Cause temporary single strand breaks in DNA
Allows free rotation of helix
Monomeric enzymes (~100kD)
Reversible catenation (knotting)
Acts by breaking phosphodiester linkage via tyrosine active site

27. Topoisomerase II (DNA gyrase)
Cause temporary double strand breaks in DNA

28. Topoisomerase II (DNA gyrase)
~ 375 kD
2 pairs of subunits: A & B
ATP hydrolysis mediated double strand break
Catenates double stranded circles
Inhibited by antibiotics