1. The Replication of DNA CHAPTER 8

2. Initiation of DNA replication Replicon : a particular origin that all the DNA is replicated from Replicator : the entire set of cis-acting DNA sequences that is sufficient to direct the inition of DNA replication, including initiator binding sites and easily unwound DNA Initiator protein : the DNA-binding protein specially recognizes a DNA element in the replicator and activates the initiation of replication The easily unwound DNA is always a stretch of AT-rich DNA

3. Initiator proteins three functions These proteins bind a specific DNA sequence within the replicator Once bound to the DNA,they frequently distort or unwind aregion of DNA adjacent to their binding sites Initiator proteins interact with additional factors required for replication initiaton

4. Examples : The E.coli initiator protein,Dna A have three different functions above. In eukaryotic cells,the initiator is a six protein complx called the origin reconnition complx (ORC). But ORC performs two of the three fuctions : binding and recriting other replication proteins to the replicator.

5. Prorein-protein and protein-DNA interactions direct the initiation process multiple DnaA-ATP proteins bind to the repeated 9- mer sequences within ORC binding of DnaA-ATP to these sequences leads to strand separation within the 13-mer repeats DNA helicases(DnaB) and the DNA helicases loader(Dnac) interactions with DonA DNA helicases loaders catalyze the opening of DNA helicases protein ring and placement of the ring around the ssDNA at the origin DNA helicases each recruit a DNA primase which synthesizes an RNA primer on each template Take E.coli for example

6. DNA helicas e DNA helicases unwind the double helix Replication in details

7. The formation of replication fork and SSBs bind to stabilise ssDNA

8. Tw decreases as DNA unwinds. Wr increases as Tw decreases. Topoisomerases remove positive supercoils.

9. DNA replication requires a RNA primer Primase make short RNA primers using ssDNA as template DNA primase is activated by interacting with the DNA helicase PRIMASE SHORT RNA PRIMERS

10. DNA polymerases catalyzes DNA synthesis

11. Sliding DNA clamps increases processivity SLIDING DNA CLAMPS Processivity : the ability of an enzyme to catalyze many reactions before releasing its substrate

12. RNAse degrades RNA base paired with DNA Removal of RNA primers leaves gaps DNA polymerase fill the gaps DNA ligase repairs the remaining nicks

13. Finishing replication Questions ? How to separate daughter DNA molecules How to copy the extreme ends of the laging strand Two solutions !!!

14. Type Ⅱ topoisomerases are required to separated daughter DNA After a circular DNA molecular is replicated, the resulting complete daughter DNA molecules remain linked to one another. Type Ⅱ topoisomerases can sepatate these DNA circles There is no inherent toplogical linkage after the replication of a linear molecule, the large size of the eukaryotic chromsomes necessitates the intrcate folding of the DNA into loops attached to a protein scaffold, and these loops must separated by topoisomerases.

15. One solution to ens replication problem Using a protein as aprimer for the last Okazaki fragment at each end of the chromosome. In this situation, the “ priming protein “ binds to the lagging strand template and uses an amino acid to provide an OH that replaces the 3’OH normally provided by an RNA primer

16. The other solution ( in most eukarotic cells ) Telomerase uses its RNA component to anneal to the 3’end of the ssDNA regin of the Telomerase Telomerase uses its reverse transcription activity to synthesize DNA to the end of RNA template Telomerase then displaces the RNA from the DNA product and rebinds at the end of the telomerase andrepeats the process

17. The telomerase regulation The proteins bound to the double-stranded regions of the telomere regulate the tolemere lengh As few telomere sequence repeat, few of these proteins will be bound to the telomere and telomerase activity will be activated As the telomere gets longer,these proteins will accmulate and inhibit the telomerase

18. Eukaryotic chromosomes are replicated exactly once per cell cycle The pr-RC formation The assembly of the pre-rc is an ordered process that is initiated by the association of the origin recognition complex with replicator Once bound to the replicator, ORC recruits at least two additional proteins, cdc6 and cdt1. These three proteins function together to recrit the putative eukaryotic DNA helicase. Pre-RC initiate the replication

19. Pre-RC regulated to allowonly a single round of replication during each cell cycle Pre-RC’s formation and activation is regulated by the Cdks Active Cdk is absent during G1, but present during s, G2 and M phases. There is only one opportunity for pre-RC to form and one opportunity to activated Pre-RCs are disassembled after they are activated or after the DNA to which they are bound is replicated.