Genetic Stability & Instability Flashcards
What consequences can recombination have?
Generates individuality
ds breaks cause rearrangements - eg. V, D, & J regions of immunoglobulins variety in antibodies
BUT also a factor in cancer formation
What are the 2 products of recombination?
Crossover & non-crossover
Occur at roughly equal frequency
What is a Holliday junction?
Crossover in inner strands of 2 dsDNA
Essential intermediate for ds breaks
How does non-homologous end-joining occur?
Nicks in strands & fragments removed Ends bound by Ku Artemis: DNA-Pkcs resects overhangs Annealing between DNA ends, Artemis: DNA-PKcs cleaves flaps Polymerase fills gaps Ligase complex ligates nicks
What is site-specific recombination?
Operate between defined sequences & mediate specific rearrangements
Eg. integration/excision of plasmid
What is the mechanism of site-specific recombination?
Sequence of site pairs up with another copy of site/similar sequence - in the middle there’s identical base sequence
Strand on each broken and covalent bond created on each
Second strand cleaved & covalent bond created
Strands separated
How can inversion systems be used?
Flagellar phase variation in Salmonella - evade immune system
Tail fibre variation in bacteriophage Mu
What is chromosome dimer resolution?
If there’s an odd number of crossovers in a circular chromosome it’ll produce dimeric chromosome that needs to be resolved
Xer-CD used at dif sites to recombine producing 2 separate circles
How does transposition work?
Find target site and cleave
Insert sequence
Gaps willed with DNA polymerase
In bacteria and eukaryotes
How are DNA double-strand breaks repaired?
2 Holliday junctions formed
Both resolved by RuvABC
DNA synthesis to regenerate DNA that’s been degraded
How are DNA single-strand gaps repaired?
RecFOR & initiation protein enables RecA to bind gap
RecA initiates strand invasion to form Holliday junction
RuvAB complex binds to junction
Junction resolved and D loop cleaved
DNA replication occurs to fill in gap
How does recombination support DNA replication?
If there’s an interruption on leading strand fork breaks
SS gap filled and stand invasion occurs
RuvAB loads onto Holliday junction
Junction resolved
This happens many times in 1 human replication cycle
How does RecA work?
Binds ssDNA helically
Increases length of DNA by 50% by stretching - viewed using cryo-microscopy
Catalyses strand-exchange, pairs with DNA strand complimentary in synapsis, and other dsDNA removed, RecA protein - needs hydrolysis of ATP
How was the polarity of RecA shown experimentally?
Circular ss-molecule (polarity 5’-3’) and ds-linear molecule
RecA protein polymerises onto circular DNA 5’-3’
ds-molecule have 1 complimentary and 1 identical strand
3 compositions of ds-molecule: non-homologous DNA at 5’ end, at both ends, or at 3’ end
All 3 catalyse synapsis
Only 1st can strand exchange occur in direction of polymerisation of RecA protein
How can base pairs be recognised in DNA stretched out by RecA?
DNA stretched between every 3 bases retaining normal spacing of bases within triplets
Ensures all triplets face out so can pair up with dsDNA bases
Transiently unwinds DNA to see if bases pair, continues until stable synaptic complex forms
What does RecBCD do?
Helicase nuclease
Recognises ends of broken chromosome & unwinds DNA strands
Digests both strands: 3’ v frequently and 5’ less frequently until reaches chi sequence
Changes activity and stops cleaving 3’ end strand and cleaves 5’ end strand more frequently
Catalyses loading of RecA protein in a similar way Okazaki fragments
What do the subunits of RecBCD do?
RecB nuclease cleaves strands at frequency determined by RecC
RecC chi scanning site jams with chi sequence & loop of DNA pushed out which is polymerised by RecA
What does RuvABC do?
RuvA stretches Holliday junction - open square planar configuration
RuvB - translocase, forms 2 rings around structure and can pump DNA away from junction
What are the 3 early models of the mechanism of recombination?
Break-join: no DNA synthesis
Copy-choice: DNA totally newly synthesised
Break-copy: part of the DNA newly synthesised
What did Weselson and Weigle discover in 1961?
Recombination requires a break in DNA
Used bacteriophage lambda with dsDNA: 1 with turbid large plaques grown with heavy isotope and 1 with clear small plaques
Bacteriophage co-infect E. coli at same time
Centrifuged progeny, top = low density, bottom = high density
For progeny with turbid small plaques expect recombination, with most DNA heavy
Means cop-choice model untrue, newly synthesised DNA would be light
What did Meselson discover in 1965?
Bacteriophage lambda with dsDNA: 1 with turbid plaques and heavy DNA, 1 with clear plaques and light DNA
Coinfect bacteria
Centrifuge progeny & plated: clear, turbid, and mottled plaques
Mottled plaques from heteroduplex DNA (DNA that don’t have same info on both strands)
What is a 2-factor cross?
1 parent mutated for a & WT for b, 1 parent mutated for b & WT for a
Progeny have parental genotypes and recombinant genotypes
Can only calculate recombination frequency if there’s reciprocality in population
What is a 3-factor cross?
Parents +,b,+ and a,+,c
Progeny has parental, single, and double recombinants
Can calculate frequency single recombinants (Rab & Rbc) and double recombinants
Can calculate predicted frequency & use to find coefficient of coincidence to find interference
What did Amati and Meselson find in 1965?
Parents +,b,+ and a,+,c BUT markers a & c can be moved further/closer to b
As distance reduced negative interference increased - if there’s a crossover there’s a higher chance of a second
How does heteroduplex DNA lead to negative interference?
Can give the appearance of 2 double recombinants from single recombination event
How can heteroduplex DNA lead to non-reciprocality?
Mismatch correction (B-b) leads to recombinant strands of genotypes - non-reciprocal
What are the parts of DNA Pol III?
Core enzyme:
- alpha, 5’-3’ polymerisation
- epsilon, proof reading exonuclease
- sigma, core assembly & stability
Gamma complex - 5 subunits, loads and unloads polymerase acts as DNA template
Beta complex - sliding clamp
Theta subunit - connects 2 pol II subunits to create dimeric polymerase
How is DNA primed?
Primer (a small RNA) supplies free 3’ OH, required by DNA pol III
Must be removed to complete replication by DNA pol I
How does elongation occur at the replication fork?
Leading strand - uninterrupted replication 5’-3’
Lagging strand - cannot replicate 3’-5’, does so in fragments individually primed by primase, produces Okazaki fragments
Lagging strands forms a loop as DNA pol III functions as a dimer
How is replication initiated at oriC?
DnaA & ATP bind - slightly opens DNA
DnaB helicase opens up ds helix with ATP
Single Stranded Binding Proteins (SSBPs) bind to DNA
DNA pol III binds and starts replication
How is replication terminated?
2 fork traps for clockwise and anticlockwise forks, if 1 is held up replication stopped there
How does replication re-start, ie. if replisome falls off?
PriA recruits PriB and DnaT to form a complex
Then DnaC and DnaB can reassemble
Why is unprotected linear DNA formed?
Replication fork collapse or replication fork renewal
How id replication repair done?
Depends on double strand breaks RecBCD produces piece of ssDNA Recruits RecA Holliday junction resolved by RuvABC PriA recognises & restarts replication
What can obstacles on DNA do?
Can lead to replication fork reversal
Produces Holliday junction, resolved by RuvABC - RuvC cuts DNA
PriA restarts replication
