L3: DNA Replication Overview Flashcards
Basics of replication
- Replication is semi-conservative (discovered by Meselson and Stahl)
- The base of the template strand must be identified and comp. base added
- Initiation, elongation and termination phase
Replication bubble (where is it established? What does it mean for directionality?
- Starting at ori, parental DNA is opened establishing 2 replication forks
- Bidirectional replication occurs, in 5’ to 3’ direction
- Continuous on leading strand but discontinuous on lagging strand
Initiation
- ori recognized by initiator proteins, which open up the double helix
- They then recruit helicases which unwind -> ssDNA
- Can’t occur de novo (must add to existing 3’ end) - requires a primer (short RNA molecule), synthesised by primase
Elongation
- Sliding clamp recruited to 3’ end of primer
- DNA polymerase associated w/ DNA via sliding clamp
- Reads bases, catalyses insertion of complementary base (5’ to 3’ i.e. adding bases to 3’ end ONLY)
- Okazaki fragments employed in lagging strand
Termination (when it occurs, overview of mechanism)
Occurs when…
- DNA polymerase encounters DNA that has been replicated
- 2 different forks meet
- fork reaches end of linear chr.
Mechanism…
- Replication complexes disassembled
- RNA primers removed and replaced w/ DNA
- DNA ligase connects adjacent strands
DNA pol requirements
- existing 3’ end (i.e. primer or newly synthesised DNA)
- dNTP (phosphates: alpha, beta, gamma starting closest to sugar)
DNA pol right hand structure
3 domains..
- Thumb (holds elongating dsDNA and maintains contact w. ss template necessary for processive synth. )
- Fingers (ssDNA wraps around, helping to position incoming nt)
- Palm (contains catalytic site for nt addition, forms a cleft where elongating DNA fits)
Main replicative polymerases, speed of replication in each group
Bacteria (E.coli)…
- 1000 nt/s
- DNA pol III (leading and lagging strand synth.)
- DNA pol I (synth of Okazaki)
Euk…
- 50 nt/s
- DNA pol ‘delta’ (lagging)
- DNA pol ‘epsilon’ (leading)
- Highly conserved
- Only 5’ to 3’ direction
- Remain attached to DNA for long stretches before dissociation (processive)
- Correct positioning only achieved w/ complementary bp
DNA pol catalytic mechanism (phosphoryl transfer rxn)
- Links 5’ phosphate of incoming nt to 3’ OH of growing DNA -> phosphodiester bond
- Nu- attack by 3’ OH on alpha-phosphate of incoming dNTP releasing 2 phosphates as pyrophosphate (hydrolysis of this provides E)
- Mg2+ ion coordinates phosphates
Specificity (Fidelity 1)
- Active site of DNA pol selective for correct base pairing
- Mismatches have a different shape
- Error rate of <1 in 100 000
- DOES NOT require energy input
Exonuclease activity (Fidelity 2)
- Replicative DNA pols have 3’ to 5’ proofreading exonuclease activity (reverse of synth) -> removes incorrectly added base
- AS of DNA pol has reduced affinity for incorrect nt; relocated to exonuclease AS which removes it, transferred back to DNA pol AS for resumed synth
- DOES require energy input
DNA pol further examples and groups
E.coli…
- pol 3 (replication)
- pol 1 (gap repair)
Euk…
- pol alpha (primase and repair)
- pol sigma (replication)
- pol epsilon (replication)
- telomerase (elongates telomeres)
- Reverse transcriptase, in contrast, uses an RNA template to synth DNA
- AS highly conserved; however, pols are grouped into families according to evolutionary lineage of rest of protein - more similarity within group than within organisms
DNA helicases (basic function, structure and defining feature in euk vs bacteria)
- Unwind dsDNA allowing replication
- Travel along strand, continuously unwinding by displacing complementary strand
- Hexameric ring protein
- Polarity defined in direction moved on strand (Bacterial move 5’ to 3’ on lagging strand whereas euk. initially wrapped around both strands until after S phase; 3’ to 5’ on leading)
E.coli DNA helicase
- DnaB helicase (homo-hexamer), loaded onto ssDNA by DNAC helicase LOADER COMPLEX
- moves 5’ to 3’ on lagging strand template
Eukaryotic DNA helicase
- Core of replicative helicase is hetero-hexameric (MCM complex; Mini Chromosome Maintenance)
- Formed from MCM2-7
- Loaded onto dsDNA in G1 phase
- Full euk. CMG (Cdc45, MCM, GINS) DNA helicase assembled and activated in S phase, transitioning to encircle ssDNA
- 3’ to 5’ on lagging strand template
