DNA Replication

1. DNA Replication • Basic vocabulary • Initiation • Replication forks • DNA polymerases • Strand differences • Ligation • Proof reading

2. Semiconservative vs conservative DNA polymerase Theta structures Replication fork Unidirectional vs bidirectional Okazaki fragments Template strand Primer Leading strand Lagging strand DNA ligase RNA primers Primase Pol 1, Pol III Processivity Nick translation Proofreading Klenow fragment Primosome DNA binding proteins 3’ and 5’ Exonucleases Replication Vocabulary

3. DNA BIOSYNTHESIS 3 Foundation Studies • Meselson and Stahl-Conservative vs Semiconservative replication • Cairns’ Replicating Fork • Okazaki’s fragments

4. CsCl 15NH4Cl Meselson and Stahl Hybrid band Observed Light 14N 14N, 15N Heavy 15N 0 0 1-3 1-3 >10 >10 Semiconservative Semiconservative Conservative

5. Leading Strand: Replication is in the direction of the moving fork “The movement of the replicating fork is unidirectional, i.e., synthesis occurs on both strands but the fork is moving in only one direction Cairns’ Replicating Fork 5’ 3’ Kornberg “DNA synthesis proceeds through the sequential addition of dNTP to the 3’ end of the growing chain of nucleotides Theta structure ERGO: Something is wrong 3’ 5’ 3’ 5’ Lagging Strand Okazaki fragments

6. CH2 B1 CH2 O B1 O O H 3’-OH O H O P O O O O O O-P-O-P-O- P-O- CH2 B2 O O O O CH2 B2 O H O H H O H Mechanism of Chain Elongation H

7. RNA Primer 3’ end 3’ OH OH DNA synthesis involves addition of dNTP to 3’ end of a as directed by a Primer Template SUMMARY OF DNA SYNTHESIS dATP, dGTP, dCTP, dTTP + Template + Primer 5’ U A G G C T A C T G A A A T C C G A T G A C T T 5’ 3’ Template

8. Stages and Events in Replication Initiation: Proteins recognize the origin Parental strands separate Primosome initiates synthesis at replication fork Elongation: Parental strands unwind at the forks Replisome complex moves along DNA Leading strand is replicated continuously Lagging stand is replicated discontinuously Termination: Duplicate chromosomes separate

9. Replication Fork

10. Loop Directionality Replication must always proceed in a 5’ to 3’ direction

11. What is a Primosome? Ans: A 600 kD protein assembly composed of at least 7 individual proteins that take part in replication What function does it perform? Ans: Primosomes conduct the initial phases of replication, They unwind the DNA, separate and keep the strands apart, lay down an RNA primer Why is it needed? Ans: Because DNA polymerase cannot initiate the synthesis of a DNA chain, and leading and lagging strands must have a 3’OH to start off and extend

12. Major Primosome Composition Primosomes assemble on single-stranded DNA in a unique region called the primosome assembly site (pas) One of the primosomes 7 proteins displaces SSB in order to localize the primosome. PriA ATP driven, displaces SSB (single strand binding protein) DnaB unwinds priming Primase An RNA polymerase (DnaG)

13. DNA Polymerases Polymerase I (low processivity) 5’-3’ exonuclease to remove RNA primer Lagging strand fill in 3’-5’ exonuclease Polymerase II DNA repair Polymerase III (high processivity) Leading and lagging strand DNA synthesis 3’-5’ exonuclease

14. Polymerase III • Mwt = 130,000 • 10 different polypeptide chains • Core polymerase = , , and  subunits • 2 molecules of  cause dimerization •  and  allow pol III to bind  subunit •  subunit forms sliding clamp that is responsible for high processivity • Assembled protein is called “holoenzyme”

15. Beta Subunit Dimer is responsible for high processivity of DNA Pol III Beta Subunit as a dimer forms a ring to clamp onto the DNA Processivity >5000 DNA

16. 3’ 5’ Template strand New strand (clips from 3’ end) 5’ 3’ Pol I, III Proof Reading Pol I only Absent in Klenow fragment

17. Bound dCTP Template 3’ end 3’exonuclease Proofreading