DNA Replication in E.

coli
Detailed Mechanism

1. Initiation
2. Elongation
3. Termination
 Initiation
• Initiation of DNA replication means primer
synthesis
• Different organisms use different
mechanisms to make primers
 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

Source: Adapted from DNA Replication, 2/e, (plate 15) by Arthur Kornberg and Tania Baker.
 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
 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
 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
 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
 The Role of the β-Subunit
• Core plus the β-subunit can replicate DNA
processively at about 1,000 nt/sec
– Dimer formed by β-subunit is ring-shaped
– Ring fits around DNA template
– Interacts with α-subunit of the core to tether
the whole polymerase and template together
• Holoenzyme stays on its template with the
β-clamp
 The Clamp Loader
• The β-subunit needs help from the γ complex to
load onto the DNA template
– This γ complex acts catalytically in forming this
processive αδβ complex
– Does not remain associated with the complex during
processive replication
• Clamp loading is an ATP-dependent process
– Energy from ATP changes conformation of the loader
so that δ-subunit binds to one of the β-subunits of the
clamp
– This binding opens the clamp and allows it to encircle
DNA
 The β Clamp and Loader

Source: Adapted from Ellison, V. and B. Source: Adapted from Henderson, D.R. and
Stillman, Opening of the clamp: An intimate T.J. Kelly, DNA polymerase III: Running
view of an ATP-driven biological machine. rings around the fork. Cell 84:6, 1996.
Cell 106(2001), p. 657, f. 3.
 Lagging Strand Synthesis
• The pol III holoenzyme is double-headed
• There are 2 core polymerases attached through
2 τ-subunits to a γ complex
– One core is responsible for continuous synthesis of
the leading strand
– Other core performs discontinuous synthesis of the
lagging strand
– The γ complex serves as a clamp loader to load the β
clamp onto a primed DNA template
– After loading, β clamp loses affinity for γ complex
instead associating with core polymerase
Simultaneous Strand Synthesis
• The γ complex and β
clamp help core
polymerase with
processive synthesis of
an Okazaki fragment
• When fragment
completed, β clamp loses
affinity for core
• Associate β clamp with γ
complex which acts to
unload clamp
• Now clamp recycles
 Lagging Strand Replication

Source: Adapted from Henderson, D.R. and T.J. Kelly, DNA polymerase III: Running rings around the
fork. Cell 84:7, 1996.
 Termination
• Bacterial replication – 2 replication forks
approach each other at the terminus
region
– Contains 22-bp terminator sites that bind
specific proteins (terminus utilization
substance, TUS)
– Replicating forks enter terminus region and
pause
– Leaves 2 daughter duplexes entangled
– Must separate or no cell division
 Decatenation: Disentangling
Daughter DNAs

• At the end of replication, circular bacterial

chromosomes form catenanes that
decatenated in a two step process
– First, remaining unreplicated double-helical
turns linking the two strands are melted
– Repair synthesis fills in the gaps