1. -General features of DNA replication in Prokaryotic
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

4. 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

5. 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

6. 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

7. 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

13. 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

14. 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

15. 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