The function of DNA is information transfer and storage

1. DNA is copied to more DNA in DNA replication

2. Gene expression i.e.

Transcription- synthesis of RNA from only one strand of a double stranded DNA helix

Translation- ribosome mediated synthesis of a polypeptide from a messenger RNA molecule

DNA replication

A polymerisation reaction with the typical 3 phases

• initiation,

• elongation,

• termination

Initiation occurs at replication origins

Initiation

• occurs at Ori C (origin of chrm replication)

• a 245 bp region with 2 conserved sequences

• 3 tandem 13bp repeats

• 4 copies of a 9 bp sequence

• Dna A protein molecules with bound ATP bind to 9 bp repeats

• Facilitated by HU protein• The 3 x 13 bp repeats are sequencially

denatured to give the open complex• A complex formed by Dna B and Dna C bind to

the melted region, Dna C is released• In the presence of SSB protein and Dna gyrase

the Dna B helicase unwinds the DNA in preparation for priming and DNA synthesis

Replication occurs at origins

Elongation

• Leading and lagging strand synthesis

• Parental DNA is unwound (helicases) and topological stress removed (gyrases/topoisomerases)

• Separated strand stabilised by SSB

• Leading strand

• Primase (DnaG protein) synthesises a short RNA primer at origin

• DNA pol III builds complimentary strand from ds primer

• continuous process

• Lagging

• Primase synthesizes many RNA primers along lagging strand

• Each primer is extended by DNA pol III

• Synthesis proceeds in 5' to 3' direction i.e. the direction opposite to the fork movement

• Synthesis is discontinuous in the form of multiple Okazaki fragments

lagging continued..........• Synthesis continues until fragment

extends as far as the primer of the previously added Okazaki sequence

• Note both strands are synthesised by a single asymetric dimer of DNA pol III that moves in the direction of the replication fork

• This is achieved in the case of the lagging strand by the DNA looping around the part of the dimer DNA pol III

• DnaB (helicase) and Dna G (primase) together form the primosome

• 1000 nucleotides of new DNA added per second to each strand

• RNA primers are removed (exonuclease activity) and replaced with DNA (polymerase) by DNA pol I and the remaining nick is sealled by a ligase using NAD as a cofactor

Termination• Eventually the 2 relication forks of E.coli meet

at a terminus region containing many copies of a 20 bp sequence called Ter

• These sequences are binding sites for a protein called Terminal utilization substance (Tus)

• Tus-Ter complex arrests the replication fork in one direction

• The other replication fork halts when they meet.

• the few hundred base pairs in between the protein complexes are replicated by an as yet unknown mechanism resulting in 2 interlinked circular chromosomes

• separation requires Topoisomerase IV• separate chrm are then segregated at

cell division

Proteins at the E coli replication Fork

SSB binds to ssDNA and

stabilizes it

DnaB protein (helicase) DNA unwinding; primosome

constituent

Dna G protein (Primase) RNA primer synthesis;

primosome constituent

DNA pol III New strand elongation

DNA pol I Excision of primers and filling

of gaps

DNA ligase Ligation

DNA gyrase supercoiling

(DNA topoisomerase II)

DNA polymerasesDNA pol I DNA pol II DNA pol III

Mol.wt.

(Daltons) 103,000 88,000 900,000

polymerizatn

rate 16-20 7 250-1000

(nucleotides/second)

3' to 5'

exonuclease

activity yes yes yes

5' to 3‘

exonuclease

activity yes no no

Functions proof reading and repair replicatn