MB-4-replication学案.ppt

2) Rolling Circle Replication Circular DNAs can replicate by a rolling circle mechanism One strand of a dsDNA is nicked and the 3’-end is extended This uses the intact DNA strand as a template The 5’-end is displaced Phage ?X174 replication cycles so that when one round is complete a full-length, single-stranded circle of DNA is released Phage l, displaced strand serves as the template for discontinuous, lagging strand synthesis Phage l Rolling Circle Model As the circle rolls right Leading strand elongates continuously Lagging strand elongates discontinuously Uses unrolled leading strand as a template RNA primers for Okazaki fragment Progeny dsDNA produced grows to many genomes before one genome worth is clipped off DNA Polymerase I Mild proteolytic treatment results in 2 polypeptides Klenow (C-terminal large fragment): 5’ ? 3’ polymerase 3’ ? 5’ exonuclease Small domain: 5’ ? 3’ exonuclease Proofreading Proofreading refers to any mechanism for correcting errors in protein or nucleic acid synthesis that involves scrutiny of individual units after they have been added to the chain. If pol I added wrong nt, won’t base pair properly Pol I pauses, exonuclease removes mispaired nt Allows replication to continue Increases fidelity of replication Klenow Fragment: 3’ ? 5’ exonuclease 5’ ? 3’ polymerase 5’?3’ exonuclease This activity allows pol I to degrade a strand ahead of advancing polymerase Removes and replaces a strand in one pass Basic functions are: Primer removal Nick translation Polymerases II and III Pol II activity is not required for DNA replication Pol II appears mostly active in repair Only pol III is required for DNA replication Pol III is the enzyme that replicates bacterial DNA Fidelity of Replication Faithful replication is essential to life DNA replication machinery has a built-in proofreading system This system requires priming Only a base-paired nucleotide can serv