分子生物学chapt21_lecture.ppt

Molecular BiologyFourth Edition Chapter 21 DNA Replication II: Detailed Mechanism 21.1 Initiation Initiation of DNA replication means primer synthesis Different organisms use different mechanisms to make primers Different phages infect E. coli using quite different primer synthesis strategies Coliphages were convenient tools to probe DNA replication as they are so simple they must rely primarily on host proteins to replicate their DNAs Priming in E. coli Primosome refers to collection of proteins needed to make primers for a given replicating DNA Primer synthesis in E. coli requires a primosome composed: DNA helicase DnaB Primase, DnaG Primosome assembly at the origin of replication, oriC uses multi-step sequence Priming at oriC Origin of Replication in E. coli Primosome assembly at oriC occurs as follows: DnaA binds to oriC at sites called dnaA boxes and cooperates with RNA polymerase and HU protein in melting a DNA region adjacent to leftmost dnaA box DnaB binds to the open complex and facilitates binding of primase to complete the primosome Primosome remains with replisome, repeatedly primes Okazaki fragment synthesis on lagging strand DnaB has a helicase activity that unwinds DNA as the replisome progresses Priming in Eukaryotes Eukaryotic replication is more complex than bacterial replication Complicating factors Bigger size of eukaryotic genomes Slower movement of replicating forks Each chromosome must have multiple origins Started study with a simple monkey virus Later consider yeast Origin of Replication in SV40 The SV40 origin of replication is adjacent to the viral transcription control region Initiation of replication depends on the viral large T antigen binding to: Region within the 64-bp ori core Two adjacent sites Exercises a helicase activity that opens up a replication bubble within the ori core Priming is carried out by a primase associated with host DNA polymerase a Origin of Replication in Yeast Yeast origins of replication are contained within