LEC 10: DNA Replication II Flashcards
Initiation of Replication on ssDNA Template
Primer Synthesis:
- dnaG synthesizes a 10-15b RNA primer on the template
Extension and Primer Removal
- The primer is extended by DNApol3 holoenzyme which synthesizes around the circle until it reaches the 5’ end of the primer. It then dissociates leaving a single strand gap.
- The primer is removed by DNA polymerase I, and the strands are sealed by DNA ligase.
Primer Removal Reaction
- DNApol1 binds to single strand break
- Primer degraded by 5’-3’ exonuclease and gap is filled by 5’-3’ DNApol activity
- DNApol1 dissociated leaving a single strand gap, which is sealed by DNA ligase
Replication of a Double Stranded Template
The initial events must involve separation of the strands so that the primers can be synthesized
DNA Supercoiling
Positively Supercoiled DNA ↓ (Topoisomerase) ↑ (DNA Gyrase) Relaxed form DNA ↓ (Topoisomerase) Negatively Supercoiled DNA
Replication Origin
Site where replication begins
– 1 in E. coli
– 1,000s in human
Strands are separated to allow replication machinery contact with the DNA
Initiation of E.Coli Replication
OriC DNA wraps around DNAa complex
- Wrapping denatures AT-rich regions of OriC
- SSB, 2x DNAb-DNAc complexes, and 2x DNAc complexes added
- Loading of DNAb helicase
Synthesis of New Strands after Initiation
The dnaG primase makes short 10-15 base RNA as it migrates along the DNA
SSB Protein
Single stranded molecules tend to fold up on themselves
• SSB is a “Helix Destabilising Protein” that melts short ds regions in ssDNA
• Builds up filament and prevents hairpin formation
Replisome Constituents
- Helicase
- Primase
- DNA polymerase III
- Sliding clamp
- SSB protein
- Topoisomerases
- DNA ligase
Topoisomerase Function in DNA Replication
- Helicase (DNAb) unwinds the DNA strands to create a replication bubble
- The unwinding creates knots in the chromosome which are resolved by DNA Gyrase (Type II Topoisomerase)
Termination of Replication
- The terminus is surrounded by Ter sites that prevent further movement of the replication fork
- The Ter sites have a 23b concensus sequence that binds the Tus protein
- Binding of Tus prevents migration of the dnaB helicase
The Eukaryotic Replisome
- Pol δ – the replicase
- Pol α/Primase – contains primase and DNA polymerase activity
- PCNA (proliferating cell nuclear antigen) – trimeric sliding clamp
- Replication Factor C (RFC) – the clamp ladder
- MCMs – helicase
- Replication Protein A (RPA) = SSB
- RNase H – nuclease that is specific for RNA in RNA/DNA hybrids
Eukaryotic Cell Cycle
• Time for DNA replication is limited (e.g. mammalian S phase lasts 6-8 hours)
• Eukaryotic forks move at only about 1/10th the rate of bacterial
• Chromosomes can be in excess of 10^8 bp
∴ Completion of replication in the allotted requires multiple origins
• In eukaryotes Origin Recognition Complex (ORC), a complex of 6 ATPases is the functional equivalent of DnaA
Core Proteins at the Replication Fork
Topoisomerases - Prevents torsion by DNA breaks
Helicases - Separates 2 strands
Primase - RNA primer synthesis
Single strand binding proteins - Prevent reannealing of single strands
DNApol - Synthesis of new strand
Tethering Protein - Stabilises polymerase
DNA Ligase - Seals nick via phosphodiester linkage
