DNA synthesis

1. DNA synthesis Kornberg detected an E. coli enzyme that incorporated radioactive dNTPs into DNA. DNA polymerase I was purified. DNA polymerase III (identified later) synthesises most of the DNA in E. coli

2. Mechanism of DNA synthesis 3’ Template strand 5’

3. Nucleotides are added to the 3’ end of the growing chain.

4. DNA polymerases synthesise DNA in the 5’ to 3’ direction absolutely require a primer Template 3’ 5’ 3’ 5’ + primer 5’ 3’ 5’ Synthesis of new DNA strand 3’ 5’ 3’ The primer can be DNA or RNA but must have a free 3’ OH.

5. “Proof-reading” or “Editing” Most DNA polymerases are multienzyme complexes that include an 3’ to 5’ exonuclease Mismatch The exonuclease excises misincorporated nucleotides.

6. Nucleotide excised by proof-reading exonuclease. The error rate in DNA replication is very low.

7. Exonuclease cuts off mismatched nucleotide Exonuclease active site Wrong nucleotide Incorporated.

8. DNA replication in E. coli The circular chromosome is 4,000,000 bp. The origin of replication oriC is 245 bp.

9. The DnaA protein binds to oriC. The DnaB and DnaC proteins bind. DnaB is a helicase that unwinds the duplex. Single-strand binding protein SSB prevents re-annealing DNA gyrase prevents supercoiling.

10. Primase joins the complex to complete the primosome. Primase synthesises short RNA primers. DNA polymerase III initiates DNA replication.

11. DNA synthesis only occurs in the 5’ to 3’ direction. One strand has to be synthesised in sections (Okazaki fragments).

12. Replicating bacterial chromosome

13. 5’ 3’ Movement of replication fork 3’ 5’ 3’ 5’ 3’ 5’ Leading strand Lagging strand [ Okazaki fragments ]

14. Simplified view of how lagging strand is synthesised. Polymerase completes Okazaki fragment and slides back to next RNA primer.

15. E. coli DNA polymerase III • 900 kDa dimeric complex • Subunit Function • catalyses phosphodiester bond formation • 3’ – 5’ proof-reading exonuclease q activates exonuclease • processivity [clamps enzyme to template] • g complex clamp loader • (5 subunits) • t dimerisation

16. The b subunit forms a sliding clamp that firmly anchors the catalytic subunit to the template.

17. Helicase Catalytic a subunit Primase b clamp Next fragment starts from here.

19. DNA polymerase I has two exonuclease activities. [Single polypeptide chain, 103 kDa monomer] Proof-reading exonuclease Second exonuclease

20. DNA polymerase I processes Okazaki fragments to complete the lagging strand. 5’ 3’ 3’ 5’ 3’ It has a 5’ - 3’ exonuclease domain that removes RNA primers. 3’ 5’ 3’ 5’ 3’ The polymerase activity replaces the RNA with DNA. Finally, DNA ligase seals the nicks.

21. DNA polymerases of E. coli DNA polymerase I II III Molecules/cell 400 100 10 Elongation rate (bases s-1 ) 20 5 1000 3’ – 5’ exonuclease + + + 5’ – 3’ exonuclease + - - Processivity 200 10,000 500,000 DNA pol III synthesises bulk DNA DNA pol II functions in DNA repair DNA pol III replaces RNA primers, also functions in repair.