We learned in chapter 20 that DNA replication is at least semidiscontinuous and requires the synthesis of primers before DNA synthesis can begin. We have also learned about some of the major proteins involved in DNA replication in E. coli. Thus, we know that DNA replication is complex and involves more than just a DNA polymerase. This chapter presents a close look at the mechanism of this process in E. coli and in eukaryotes. We will look at the three stages of replication-initiation, elongation, and termination-in a variety of systems. | Molecular Biology Fourth Edition Chapter 21 DNA Replication II: Detailed Mechanism Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 21- 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 21- Priming at oriC Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker. 21- . | Molecular Biology Fourth Edition Chapter 21 DNA Replication II: Detailed Mechanism Lecture PowerPoint to accompany Robert F. Weaver Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 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 21- 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 21- Priming at oriC Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker. 21- 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 21- 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 21- 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 .
