DNA replication

Dna replication

OriC

- 5 binding sites (R1, R2, R3, R4, R5, sites rich in A & T) for key initiator proteins (DnaA) - Region rich in A=T (DNA unwinding element, DUE) - 3 additional Dna-binding sites (I1, I2, I3) (only when DnaA is complexed with ATP) - Binding sites for FIS & IHF proteins

IMPORTANT: DnaA is crucial for initiation (AAA+ ATPase); its active form is bound to ATP, the inactive one to ADP (ATP is slowly hydrolysed to ADP).

Initiation

• 8 DnaA proteins assemble on R & I sites on OriC.
• Dna wraps around complex (right-handed helix); this favours opening at DUE region (IHF, HU, FIS stabilize loop).
• DnaC (AAA+ ATPase) loads DnaB helicase (hexamer) at DUE. DnaC-DnaB complex activates DnaB helicase.
• DnaC hydrolyses ATP and detaches, leaving DnaB to open the replication fork and migrate along in the 5’→3’ direction.
• Dna polymerase III binds to DnaB + SSB proteins bind to strands to stabilize + Dna Gyrase relieves topological pressure.

IMPORTANT: Initiation is the only phase known to be regulated. Dna pol III binding to Dna signals completion of initiation.

Hda (AAA+ ATPase) protein binds to Dna pol III & interacts with DnaA ATP hyd. to ADP. DnaA complex disassembles at origin.

Position of adenine (palindromic sequence: (5’)GATC)6. DAM Methylase methylates N after replication. Dna is hemi-methylated and it sequesters to the plasma membrane and binds to SeqA proteins. Entire DNA needs to be methylated before it can begin another round of replication.

Elongation

• Primase (DnaG) synthesises primer (RNA) on leading strand (primosome = DnaB (helicase) + DnaG (primase)).
• Leading strand: Dna pol III adds deoxyribonucleotides (Dna pol III linked to DnaB helicase on opposite strand leading strand synthesis proceeds continuously).
• Lagging strand: Primase synthesises RNA primer and Dna pol III adds deoxyribonucleotides. For synthesis to occur on both strands, the lagging strand must loop so that the 3 subunits of Dna pol III can function (1 subunit on leading and 2 cycle continuously on lagging).
• DnaB helicase moves along and unwraps replication fork; DnaG primase occasionally associates and forms RNA primers. Dna pol III positions new beta sliding clamp; clamp-loading complex is an AAA+ ATPase.
• Once an Okazaki fragment has been completed, its primer is removed by Dna pol III and replaced. The nick is sealed by Dna Ligase.

Termination

• TER sequence signals the end of replication; binding sites for TUS protein replication fork arrested in one direction, other fork halts when it meets the first.
• Topoisomerase IV separates the two circular chromosomes.