bacterial DNA replication

semi-conservative DNA replication

• one round of replication produces 2 hybrid molecules

• second round of replication produces half hybrid half new DNA molecules

• one strand used as template for making new ones

bidirectional replication

• large linear chromosomes have too much DNA to be replicated from a single origin

• replication begins at origin

• DNA begins to unwind at origin so replication bubble produced by unwinding of double helix

• 2 replication forks at each end of the replication bubble

• forks proceed outwards in both directions unwinding and replicating DNA until they meet

replicon

segment of DNA that undergoes replication

rolling circle replication

• initiated by break in one of the nucleotide strands so the 3’ OH group and 5’ phosphate group are exposed

• new nucleotides added at 3’ end using unbroken strand as a template

• as nucleotides added the 5’ end of the broken strand is displaced from the template strand

• 3’ end grows around the circle

replication fork

• leading strand - moves towards opening of the replication fork, 5’ to 3’ continuous replication as strand in 5’ to 3’ direction

• lagging strand - moves away from opening of replication, 3’ to 5’ discontinuous replication as a result of antiparallel nucleotide strands

• 2 units of DNA polymerase III present at replication fork - one for leading strand, one for lagging strand

DNA synthesis

• insertion site - where nucleotide addition occurs

• primase produces primers (small section of RNA) which have 3’ hydroxyl group 

• 3’ OH group attacks 5’ phosphate group of incoming dNTP so that nucleotides joined one at a time to 3’ end of newly synthesized strand

• lagging strand - new primer generated for each Okazaki fragment

DNA polymerase I

• corrects mistakes in DNA replication as has 5’ to 3’ exonuclease activity

• proofreading - one base mispaired with another, polymerase repositions mispaired 3’ terminus into the 3’ to 5’ exonuclease site, exonuclease hydrolyses mispaired base

• mismatch repair - any incorrectly paired bases produce deformity in secondary structure, deformity recognized by enzymes which remove the incorrect nucleotide and use the nucleotide strand as a template so it can be replaced

primer removal

• DNA polymerase I 5’-3’ exonuclease responsible for removing RNA primers and replacing them with DNA nucleotides

• this leaves behind ‘nick’ where 3’ OH group of newly synthesized DNA molecule not connected to 5’ phosphate group of adjacent nucleotide - DNA ligase forms phosphodiester bond

DnaB helicase

• 5’ to 3’ helicase

• unwinds parental DNA

• homohexamer - forms ring which gives it high processivity

• tracks along lagging strand

DnaG primase

• binds to DnaB helicase - in correct position for RNA primer synthesis

• synthesizes RNA primers at origin of replication

• binding to DnaB helicase stimulates activity

origin of replication

• DNA synthesis starts with short RNA primer - 2 bidirectional replication forks formed 

• DnaA binds to oriC and causes short section of DNA to unwind - allows helicase and other single-strand binding proteins to attach to the polynucleotide strand

• DNA unwinding element - site of where DNA opened for replication, AT rich

• IHF and Fis - DNA bending proteins which aid the initiation reaction

DnaB and DnaC

• DnaB loaded at fork by DnaC

• DnaC-ATP ring binds to DnaB ring and opens it for loading

• DnaBC complex binds to DnaA which causes 2 DnaB rings to be produced - one for each replication fork

• ATP hydrolysis releases DnaC so DnaB is left bound to DNA

accessory proteins - SSB

• attach to exposed single-stranded DNA to protect single-stranded nucleotide chains

• prevent formation of hairpins - base pairs on same strand line up due to sequence homology

• form tetramers which cover 35-65 nucleotides

DNA gyrase

• topoisomerase - control supercoiling of DNA

• type 2 topoisomerase - creates double-strand breaks

• releases tension of supercoil by binding to 2 sections of DNA, cuts strand that’s irregularly bound (makes double-strand break in segment of DNA helix) and passes other strand through before resealing strand

• this removes twists in DNA to reduce supercoiling

DNA polymerase III

• main workhorse of replication

• highly processive - able to add lots of nucleotides without releasing the template strand

• adds nucleotides at 3’ end of growing strand - 5’ to 3’ adds nucleotides

• 3’ to 5’ corrects errors

clamp loader and beta sliding clamp

• 2 polymerases - alpha subunits, synthesize DNA on leading and lagging strands

• gamma complex - loads beta clamp onto lagging strands, opens beta clamp and slots it onto where RNA primer is synthesized - multiple loaded onto Okazaki fragments

• tao (part of gamma complex) holds structure together

• beta clamp - homodimer

• beta clamp encircles primer that synthesizes DNA - allows DNA polymerase to slide easily along the template strand during replication

• binding to ATP allows opening of the clamp, ATP hydrolysis allows clamp release and ring closure