Chapter 21 DNA Replication II: Detailed Mechanism Objectives -General features of DNA replication in...

Chapter 21DNA 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• The pol III holoenzyme synthesizes DNAat the rate of about 730 nt/sec in vitro• The rate in vivo is almost 1000 nt/sec• This enzyme is highly processive both invitro and in vivo

21.1 Initiation• Initiation of DNA replication means primersynthesis• Different organisms use differentmechanisms to make primers

• Different phages infect E. coli using quitedifferent primer synthesis strategies• Coliphages were convenient tools to probeDNA replication as they are so simple theymust rely primarily on host proteins to

Origin of Replication in E. coliPrimosome assembly at oriC occurs as follows:– DnaA binds to oriC at sites called dnaA boxesand cooperates with DNA polymerase and HUprotein in melting a DNA region adjacent toleftmost dnaA box– DnaB binds to the open complex and facilitatesbinding of primase to complete the primosome–primes Okazaki fragment synthesis on laggingstrand– DnaB has a helicase activity that unwinds DNAas the replisome progresse

Priming in E. coli• Primosome refers to collection of proteinsneeded 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 ofreplication, oriC uses multi-step sequence

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

Elongation• Once a primer is in place, real DNAsynthesis can begin • An elegant method of coordinating the synthesis of lagging and leading strands

-keep the pol III holoenzyme engaged withthe template • Replication can be highly processive andso very rapid

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 thetemplate 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