DNA replication in prokaryotes

DNA replication

A semi-conservative process where each daughter DNA molecule contains one parental strand and one newly synthesised strand

Semi-conservative replication

Parental DNA strands separate and each serves as a template for a new strand

Replication fork

Y-shaped structure where DNA is unwound and replicated

Stages of DNA replication

Initiation, elongation, termination

Origin of replication (oriC)

Specific DNA sequence where replication begins in E. coli

Bidirectional replication

Replication proceeds in both directions from the origin, forming two replication forks

Semi-discontinuous replication

Leading strand is continuous, lagging strand is discontinuous

Leading strand

DNA strand synthesised continuously in the 5′→3′ direction

Lagging strand

DNA strand synthesised discontinuously as Okazaki fragments

Okazaki fragments

Short DNA fragments synthesised on the lagging strand

RNA primer

Short RNA sequence required to initiate DNA synthesis

Direction of DNA synthesis

Always 5′→3′

DNA ligase

Enzyme that joins Okazaki fragments after primer removal

DNA helicase (DnaB)

Unwinds DNA at the replication fork using ATP

DnaB movement

Moves 5′→3′ on the lagging strand template

DnaC

Loads DnaB helicase onto DNA at the origin

DNA gyrase

Type II topoisomerase that removes positive supercoils during replication

Positive supercoiling

Overwinding of DNA ahead of the replication fork

Type II topoisomerase

Breaks both DNA strands and requires ATP

Type I topoisomerase

Breaks one DNA strand and does not require ATP

Single-strand binding protein (SSB)

Prevents reannealing and protects ssDNA

SSB function

Promotes DNA unwinding and prevents nuclease attack

DNA primase (DnaG)

RNA polymerase that synthesises RNA primers

Primase binding

Associates with DnaB helicase at the replication fork

DNA polymerase III

Main replicative polymerase in E. coli

Pol III holoenzyme

Asymmetric dimer coordinating leading and lagging strand synthesis

β-clamp

Sliding clamp that increases DNA polymerase III processivity

β-clamp structure

Ring-shaped dimer with six globular domains

Clamp loader (γ-complex)

Loads the β-clamp onto DNA

PCNA

Eukaryotic equivalent of the β-clamp

Lagging strand loop

Allows coordinated synthesis with the leading strand

DNA polymerase I

Removes RNA primers and replaces them with DNA

Pol I 5′→3′ exonuclease

Removes RNA primers

Pol I 3′→5′ exonuclease

Proofreading activity

Pol I 5′→3′ polymerase

Fills gaps after primer removal

Klenow fragment

Pol I fragment lacking 5′→3′ exonuclease activity

DNA ligase cofactor in E. coli

NAD⁺

DNA ligase function

Forms phosphodiester bonds between adjacent DNA fragments

E. coli chromosome replication time

Approximately 40 minutes

Minimum E. coli division time

Approximately 20 minutes in culture

Multiple replication origins

Result from re-initiation before prior replication finishes

Initiator protein

DnaA

DnaA function

Binds oriC and initiates DNA replication

DnaA activity depends on

ATP binding

oriC size

Approximately 245 base pairs

oriC features

AT-rich 13-mers, DnaA 9-mer binding sites, GATC sites

GATC sites

Targets for Dam methylase

Hemimethylated DNA

Newly replicated DNA methylated on only one strand

OriC sequestration

Prevents premature re-initiation of replication

Membrane-bound proteins

Bind hemimethylated oriC for ~10 minutes

Reactivation of DnaA

Conversion of DnaA-ADP to DnaA-ATP

Additional E. coli DNA polymerases

Involved in DNA repair and damage tolerance