Lec 16 - DNA Replication, Repair, & Recombination Flashcards
DNA polymerase - nucleotide incorporation
which nuc used? how is it stabilized? processive vs distributive?
- extends growing primer strand on 3’ end w complemetary dNTP
- Two Mg2+ ions coordinate 3’-OH nucleophile
- 3’-OH is required (made more powerful nuc by Mg2+)
- Processivity is the # of cycles before polymerase dissociates (opposite is distributive)
DNA pol translocates 1 nucleotide after dNTP addition (sometimes dissociates/falls off instead)
DNA polymerase - fidelity
first check of fidelity? error rate?
- first check for fidelity is active site geometry: steric constraints disallow energetically favorable mis-pairing
- error rate 1 in 10,000 - 100,000 bases
(e coli genome is 4 mill bp… 4 mill/10k & 4 mill/100k –> 40-400)
DNA polymerase - proofreading
proofreading improves fidelity how? by what rate? final error rate?
- proofreading catches errors after incorporation, befoore translocation
- cleaves of mispaired base, tries again
- improves fidelity 10^2 to 10^3
- final error rate 10^-6 to 10^-8 (normal error rate 10^-4 - 10^-5) improves fidelity 100-1000 times)
(E coli replisome)
E coli DNA Polymerase I, III
pol I vs pol III? speed? replicative DNA helicase? couples what 2 things
DNA pol I
- first polymerase isolated
- abundant, single subunit; too slow, non-processive
- two exonuclease domains responsible for DNA cleanup (“nick translation”)
DNA pol III
- fast (1000 bp/sec); high processivity (>500,000 bp); clamped on substrate
- beta sliding clamp locked to helix (symmetric dimer)
- 3 pol III cores
- replicative helicase ahead polymerase separating two bp strands needed to copy (hexamer that forms ring around one DNA strands; energetically couples unfav sep of DNA w/ATP hydrolysis)
replication fork
- leading strand synthesized 5’ to 3’ as fork moves
- lagging strand synthesized in 5’ to 3’ in opp dir of leading; synthesized discontinuosly via Okazaki fragments
Primase synthesizes RNA primers
initial problem? solution? new problem? solution?
- problem: DNA polymerase can only extend
- solution: primase synthesizes RNA primer that DNA Pol III extends with DNA
- new problem –> DNA full of short RNA primers
finishing the lagging strand
- DNA pol I removes RNA primer and replaces with DNA with “nick translation” (via 5’ to 3’ exonuclease activity)
- DNA ligase seals remaining DNA-DNA “nick”
Bacterial Replication Machinery
- SSB
- DnaB protein (helicase)
- primase (DnaG protein)
- DNA pol III
- DNA pol I
- DNA ligase
- DNA gyrase (DNA top II)
Single-stranded Binding protein SSB
- coats lagging strand until its replicated
- binding to ssDNA
DnaB protein (helicase)
- DNA unwrithing as it untwists; primosome constituent
Primase (DnaG protein)
- RNA primer synthesis; primosome constituent
DNA pol III
- new strand elongation
DNA pol I
- filling gaps; excision of primers
DNA ligase
- ligation
DNA gyrase (DNA top II)
- supercoiling: negative supercoil promotes unwinding
(similar to bacteria, more complex)
Eukaryotic replisome
2 replicative polymerases? clamp? helicase complex?
- leading strand synthesized by elipson ε DNA polymerase, lagging by delta δ DNA polymerase
- Processivity clamp PCNA (proliferating cell nuclear antigen) acts same way as clamp
- PCNA clamp is trimer (instead of beta dimer clamp); 6 proteins that are diff instead of same
- replicative helicase has MCM2-7 complex
coordinating replication
Eukaryotic replication initiation + end replication problem
each origin replicates exactly once; persistent replication bubbles must be avoided somehow…
Initiation
- ORC (origin recognition complex) is an ATP dependent helicase loader that binds to origin/ATP during G1 (fires only in S, once)
- MCM2-7 is a replicative helicase
End problem
- because the linear chromosomes, RNA primers at extreme 5’ end cant be fixed, leaving gap behind after primer removed (lost genetic info)
Bacterial vs Eukaryotic genome replication
chromosome shape? origin(s) of replication?
Bacterial:
- large circular chromosome
- two strands copies together, no single-stranded DNA region
- initiation at a single origin of replication
- elongagtion at two replication forks traveling around circle
- each fork replicates >2 mil bps
Eukaryotic:
- long linear chromosomes
- multiple origins of replication
- forks create replication bubble (2 replicating duplexes in bubble & 1 parental duplex outside); when forks meet, they collide and dissasemble
- replication initiation & end replication problem…
telomeres + telomerase
telomeres
- sequences at end of eukaryotic chromosomes
- repetitive buffer sequences with no genetic info; eventually eroded
- puts limit on replication & plays a role in aging
telomerase
- reverse transcriptase (RNA template–> DNA)
- expressed in germline; if reactivated in somatic cells, can lead to cancer
DNA replication is semiconservative (1 old, 1 new strand)
Meselsohn-Stahl experiment
metabolic labeling
- grown in heavy nitrogen –> 1 band, all heavy
- grown in light nitrogen –> 1 band, medium (new DNA light, parent DNA heavy)
- light nitrogen (gen 2) –> 2 bands (daughter duplex splits to 1 heavy, 1 light.. replicated strand is light.. results in medium weight band and completeley light band)
cells grown in heavy N isotope and then light one, to track duplex in E
PCR - polymerase chain reaction
amplifies –> 1 billion copies of target DNA
- 2 short primers, 1 at end of each target; 3’ end facing inward
- (1) heat to denature/melt/separate strands to form ssDNA
- (2) cool to anneal primers onto DNA template
- (3) DNA polymerase extends primers from 3’ end
- (4) cycling (repeat process); DNA polymerase must be thermostable; PCR products double each time, exponentially
DNA polymerase requires a primer
DNA legions
3 examples
if not repaired, legions become mutations
DNA legions
- methylation induced mispairing (methylated G with T)
- loss of base
- UV induced pyrimidine dimer (kink)
direct repair
-O6-methylguanine DNA methyltransferase transfers 1 methyl off guanine onto cycteine (cannot be recycled)
cell makes entire protein used in reversing 1 mutagenic change
