DNA replication

1. DNA replication Learning objectives Understand the basic rules governing DNA replication Introduce proteins that are typically involved in generalised replication Presented by Aparna Microbiology Faculty .

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

10. Meselson-Stahl experiments confirm semiconservative replication

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

12. Starts at origin Initiator proteins identify specific base sequences on DNA Prokaryotes – single ori site E.gE.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

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

20. RNA primers required

21. Core proteins at the replication fork - 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 Topoisomerases Helicases Single strand binding proteins Primase DNA polymerase Tethering protein DNA ligase

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

25. Core proteins at the replication fork Nature (2003) vol 421,pp431-435 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

29. `Thank you