proteins in dna replication

what proteins are involved in dna replication in prokaryotes?

DnaA

DnaB

DnaC

primase

DNA polymerase III holoenzyme

function of DnaA

activates initiation of replication

• regulated by ATP

• binds to double stranded 9mer regions

• operates in hexameric complex

function of DnaB

DNA helicase

• inactive when bound to the helicase loader

• further unwinds DNA, extending the replication fork/bubble

• helicase recruits primase which causes helicase to dissociate

function of DnaC

helicase loader

function of primase

synthesises small stretch of RNA nucleotides

DNA polymerase III holoenzyme can only bind to 3’ hydroxyl group

allows DNA synthesis to begin

function of DNA polymerase III holoenzyme

contains everything required for the synthesis of DNA

• contains sliding clamp and sliding clamp loader

what proteins are involved in DNA replication in eukaryotes

• helicase

• DNA primase

• DNA polymerase (⍺, δ, ε)

• DNA ligase

function of helicase

unwinds parent DNA strands

hexameric protein

uses energy from ATP hydrolysis

functon of DNA primase

adds short, 12bp RNA primer with 3’ hydroxyl group for DNA polymerase δ to attach to

function of DNA polymerase ⍺

extends RNA primer with deoxyribonucleosides for another 25 nucleotides

produces RNA-DNA primer

function of DNA polymerase δ

extends RNA/DNA primer following sequence of template strand (DNA replication)

occurs in 5’ to 3’ direction

DNA polymerase δ has high fidelity - only base pairs with exact geometry fit in active site

function of DNA polymerase ε

synthesises leading strand of DNA in 5’ to 3’ direction

function of DNA ligase

join together fragments of DNA sequences

what is the leading strand?

DNA strand that is synthesised in the same direction as the replication fork, 5’ to 3’

what is the lagging strand?

synthesised discontinuously

DNA polymerase only functions 5’ to 3’ but lagging strand runs 3’ to 5’

RNA primers every 100-200 nucleotides

elongation of primers produces multiple okazaki fragments (mostly by DNA polymerase δ)

fragments joined together by DNA ligase

how is dna proofread?

DNA polymerase proofreads DNA using 3’-5’ exonuclease activity

incorrect base is sensed by physical distortion

polymerase has exonuclease domain that can remove incorrect base

DNA helicase hexameric structure

arrangement of 6 identical subunits

3 different states that interconvert during ATP hydrolysis, driving movement of helicase

2x subunites more likely to bind ATP, 2x likely to bind ADP + Pi, 2x empty subunits

what are topoisomerases

help to releive tension in DNA due to supercoils that are formed during replication

what are supercoils?

as DNA strands are separated, tension builds in the remaining DNA helix as the strands twist around each other more and more

positive vs negative supercoils

positive = twisted

negative = not twisted

function of topoisomerase I

binds to DNA at random points and breaks a phosphodiester bond in ONE strand

function of topoisomerase II?

makes breaks in both strands then religates them