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Chapter 21 DNA Replication II: Detailed Mechanism Objectives

Chapter 21 DNA Replication II: Detailed Mechanism Objectives -General features of DNA replication in Prokaryotic -Enzymology of DNA replication -DNA Replication : Detailed mechanisms -Speed of replication -initiation -Elongation. Speed of Replication

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Chapter 21 DNA Replication II: Detailed Mechanism Objectives

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  1. Chapter 21 DNA Replication II: Detailed Mechanism Objectives -General features of DNA replication in Prokaryotic -Enzymology of DNA replication -DNA Replication : Detailed mechanisms -Speed of replication -initiation -Elongation

  2. Speed of Replication • The pol III holoenzyme synthesizes DNA at the rate of about 730 nt/sec in vitro • The rate in vivo is almost 1000 nt/sec • This enzyme is highly processive both in vitro and in vivo

  3. 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

  4. 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 DNA 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 –primes Okazaki fragment synthesis on lagging strand – DnaB has a helicase activity that unwinds DNA as the replisome progresse

  5. 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

  6. Summary: The yeast Ori contained with autonomously replicating sequence (ARSs) That composed of 4 important regions: A, B1 ,B2 and B3: A is 15 bp long( 11 bp consensus conserved ARSs) B3: allows DNA bending

  7. Elongation • Once a primer is in place, real DNA synthesis can begin • An elegant method of coordinating the synthesis of lagging and leading strands -keep the pol III holoenzyme engaged with the template • Replication can be highly processive and so very rapid

  8. The Pol III Holoenzyme and Processivity of Replication • Pol III core alone is a very poor polymerase, after assembling 10 nt it falls off the template • Takes about 1 minute to reassociate with the template and nascent DNA strand • Something is missing from the core enzyme – The agent that confers processivity on holoenzyme allows it to remain engaged with the template – Processivity agent is a “sliding clamp”, the β- subunit of the holoenzyme

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