homologous recombination Flashcards
two pathways of HR?
RecFOR - ss gaps
RecBCD - ds breaks
when does cell do HR?
DNA ds break
dsDNA fragment with homology to area on chromosome present and integrated
DNA ss gap
ss gap repair
RecFOR (3 different proteins)
Loads RecA onto ss gap
strand invasion of homologous chromosome
forms holliday junction - covered in RuvA and RuvB
cut by RuvC
leads to repair of full chromosome
ds break repair?
2 ends of strand at break recognised by RecBCD complex (B does recognition)
RecB loads on DNA and forms ss region at Chi site (helicase and nuclease activity) [End processing]
Rec B and D (helicase) action means that before chi recognition DNA is open
ss region at chi site recognised by Rec C and RecBCD activity changes (rec B opens DNA just a bit now and D no longer works)
then ss region is covered by RecA which identifies homologous region on other chromosome [Strand invasion]
synapsis and strand exchange involving hollidat junction and loading of RuvABC resolvases [holliday junction resolution]
5 mechanisms of genetic recombination?
Homologous recombination
non homologous end joining
transposition
site specific recombination
independent assortment of chromosomes in meiosis
first 4 affect chromosome integrity and directly affect genome stability
aspects of HR?
faithful pathway of repairing DNA damage when both DNA molecules are cleaved at similar location
what resolves holliday junctions?
in strand invasion - new DNA copied from homologous region of other chromosome
cleavage by resolvase
RuvABC
(two ds strands next to each other, 2 crossovers between strands on inside)
both outer strands cleaved or both inner strands cleaved
=non crossover
small sequence of DNA is copied from intact homologous DNA to repair broken DNA - so can transfer some different markers by copying them
region that was from intact chromosome goes to damaged one
copied region
other copied region goes to the intact homologous chromosome
just change strands at cleaved point to give product
both outer strands cleaved = no cross over - some exchange but not connected
one inner and one outer cleaved = cross over
Blue molecule before - black after where junction was
vice versa for other
IDK you need to look at diagrams…
non homologous end joining?
mechanism for repairing ds breaks
not as faithful as HR
can be carried out when no homologous region exists to copy from
takes place during most of eukaryotic cell cycle as there is no 2nd copy of DNA from s phase
important source of genetic diversity, e.g. Ab diversity generation
non homologous end joining process
Ds break ends detected and protected by Ku (also holds ends near each other)
sometimes ends can be directly ligated but a lot of the time end processing is needed
done by various enzymes:
adding nucleotides - polymerase
removing nucleotides - nuclease
modifying nt - kinase, phosphatase, phosphodiesterase
changes original DNA sequence and therefore creates mutation
Dna ligase 4 ligates ends together
strand invasion?
one strand of each ds end of break goes to one strand of homologous region
(need to look at diagram to see properly)
initially forms 3 stranded intermediate with duplex dna of homologous region
non base paired strand of duplex kicked off (branch formation)
ss nucleoprotein strand then moves along homologous region and binds more of it
moves branch point further down kicking off more of other strand (branch migration)
this extends the length of RecA nucleoprotein bound to homologous region
you can see how Holliday junctions are formed from here (see diagrams and try draw it more words won’t help)
site specific recombination examples?
e coli chromosome dimer resolution
bacteriophage lambda integration and excision
flagellar phase variation in salmonella
bacteriophage Mu tail fibre variation
SSR properties?
recombinases interact at specific target sites
ds cleavage
religation
no loss or gain of nucleotides
no DNA synthesis
results in genetic recombination
nature of change depends on orientation of target sites:
- head to head
-heat to tail
(imagine arrows pointing at each other or in same direction)
SSR inversion consequence?
Head to head target sites
parts of target sites exchanged in process
DNA loops so both target sites align in same direction (recombinase puts target sites together)
recombination between the sites
sequence between inverted
exchange between target sites next between recombination sites
(draw it)
SSR deletion/integration consequence
head to tail target sites
DNA loop so target sites align (recombinase places sites together)
recombination between them
causes either DNA to be excised as circular molecule or circular molecule with target site to be integrated
same sort of logic as other one
(draw it)
E. coli chromo dimer resolution
repair of ds break by Hr can lead to serious problem with circular chromosome
remember if inner and outer site each cleaved then one molecule before site, otehr one after
for circular chromosomes this results in them being dimerised
occurs when when Ds break happens during DNA replication
HR between homologous chromosomes while theyre being replicated (see diagram)
can end up with no crossover and 2 separate molecules after replication or crossover forming dimer
(see a diagram)
head to head sites in terminus dif region
XerXD rexombinase carries it out
formation of new crossover
allows separation of two linked chromosomes
exchange of DNA between the two
(see diagram)
integration of lambda genome into e coli chromosome?
head to head crossover sites in lambda genome
attP locus
head to head target sites in e coli chromosome
attB locus
lambda uses Int (integrase) protein
composite target sites created called attL and attR (left and right)
head to head on same genome - but line up with their corresponding site on the other organism’s genome
(see diagram)
bacteriophage mu tail fibre variation?
SSR using inversion system
head to head target sites
first two parts of tail -
constant part encoded by Sc gene, outwith target sites
variable part -
encoded by Sv and U
alternative proteins encoded by Sv’ and U’
gin region encodes recombinase outwith other side of sites that causes inversion when expressed
inversion by head to head sites reverses variable region around
either
ScSvU
ScSv’U’
RecBCD subunit function
3 subunits
RecC chi scanning site
RecB - 2 parts - helicase and nuclease - open and cut
RecD - only helicase
RecFOR and RecA function
RecFOR recognises ss gap in DNA
ss gap usually bound by SSB protein
RecFOR loads RecA onto the ss region replacing SSB
initially slow nucleation step so just a few at start
once a few molecules loaded, quicker to load rest
RecA can extend the nucleoprotein filament a small bit into the DS region after stramd
forms nucleoprotein filament with ssDNA region
homology search and strand invasion by RecA nucleoprotein filament
searches for homology and invades homologous DNA sequence
forms a D loop (see diagram)
DNA synthesis initiated from invaded strand as a template
continues and reaches recessed broken part of other end of break
on both sides
how does recA find homology?
RecA catalyses short contacts between ss nucleoprotein filament with homologous chromosome in random manner
can do intersegmental sampling where it samples 2 parts of chromo at once on other sides of folds
also can slide along
all contacts short lived until homologous region found
base pairing can happen between ssDNA and one of strands of homologous region
forms D loop
RuvABC finction in HJ resolving
DNA synthesis from nucleoprotein filament along homologous chromosome forms the cross looking junctions (diagram :))))
after dna synth complete and sna molecules joined by HJ
resolved by RuvABC
each of two HJ recognised by RuvA - binds and keeps them in open conformation
RuvA can load RuvB
B can migrate HJs in ATP dependent manner
can move to either side
responsible for branch migration
RuvC - endonuclease - does cleaving at HJs (see earlier)
nicks are ligated by replicative ligase
useful diagram in pt3 of HR biochem lecture
