Recombination 2 Flashcards
1
Q
What are three basic requirements for recombination to occur?
A
- One of the DNA molecules must have a single-stranded region ‘
- One of the molecules must have a free 3’ end
- The single stranded region and the 3’ end must be located in a region of common homology
** no ds break
2
Q
RecD
A
the faster helicase that moves from the 5’ end to the 3’ end
3
Q
RecB
A
both a helicase moving 3’ to 5’ and an endonuclease for both strands
4
Q
RecC
A
threads each strand through a tunnel and recognizes the chi site
5
Q
RecB
A
stops cutting the strand with the Chi site at the 3’ terminus and binds to RecA monomers and loads them onto the chi strand (3’ terminus)
6
Q
RecA
A
polymerizes in the 5’ to 3’ direction on the chi site
7
Q
What are the three stages of RecA participation in strand exchange?
A
- presynapsis
- synapsis
- postsynapsis
8
Q
What is the meaning of “extended” DNA form?
A
- In stead of 1 turn every 10.5 bases it has one turn every 18.6 nucleotides
- In spiral form but ss stranded but is a spiral due to recA filament
Longer
9
Q
what is the minimum length of homology for recombination to occur
A
50 bp
10
Q
What eukaryotic complex performs a similar function to RecBDC in bacteria?
A
MNR
11
Q
where is the chi site and which subunit of RecBCD recognizes it?
A
- Sequence found on the strand that terminates with the 3’ terminus
- RecC
12
Q
Rec- mutants
A
all have the same phenotype, unable to support generalized recombination
- 10-20 loci discovered in E. coli
13
Q
when do bacteria participate in recombination?
A
- bacteria do not generally exchange large amounts of duplex DNA but will occasionally under there conditions:
- free 3’ end of ss from conjugation
- ss gaps from radiation damage
- phage rolling circle replication
- repair of a lesion in DNA replication
14
Q
what must occur for recombination between 2 ds DNAs?
A
a 3’ end must be generated (conjugation)
15
Q
chi site
A
- RecBCD system is stimulated by chi sequences
- only read from 3’ strand so orientation is important because it only functions in one direction
- occurs every 5-10 kb in the E. coli genome (1,000 sites per genome)
16
Q
what are the activities of the RecBCD complex?
A
nuclease and helicase
17
Q
what is the mechanism of RecBCD?
A
- RecBCD joins to the end of a ds break
- it translocates using the two helicases RecB/D
- RecB will accumulate in a loop of DNA infornt when it hits a chi site
- RecC recognizes the chi site and changes conformation, Rec C signals RecD to stop, and when stopped
- RecD signals to RecB to cut DNA because it is also a nuclease; RecB is on a flexible tether to swing back and forth, but is closer to the 3’ end, which causes the nuclease to chop the 3’ end more frequently, which is what reacquires the RecC and RecD signals
- RecB cuts where it is and continues unwinding DNA, loading RecA
- RecB recruits RecA to the single-stranded DNA at the 3’ end
18
Q
in the RecBCD complex, which helicase is fast and which is slow?
A
RecB is the slow helicase and RecD is the fast helicase
19
Q
pre-synapsis
A
- ssDNA is coated with RecA and can synthesize everything except the DNA duplexes, so SSB proteins are needed to resolve those duplexes to mediate RecA to finish
- Coat and compete off SSB
- Ss circle is fully coated with RecA
20
Q
how is RecA able to find the region of homology between two molecules
A
presynapsis complex
21
Q
what two molecules achieve strand invasion?
A
RecA and SSBP
22
Q
synapsis
A
- Scanning and alignment
- ssDNA coated with RecA
- RecA is scanning dsDNA
- When it finds common homology, it forms two kinds of associations, starting as a paranemic joint and then into a plectonemic joint
- fastest step
23
Q
postsynapsis
A
- Replacement of the displaced strand
- RecA works around entire molecule and replacing strand in the duplex with the heteroduplex
24
Q
What are the three stages of RecA participation in strand exchange?
A
- presynapsis: slow, RecA and SSB coat ssDNA, extends helix
- synapsis: fast, alignment only of complementary sequences in ssDNA and dsDNA that participate in strand exchange
- postsynapsis: strand exchange, slow, ssDNA replaces on strand in dsDNA with the two DNAs that are intertwined in a join molecule intermediate
25
what is the RecA protein structure?
- Forms a spiral before DNA is bound to it
- Stretches out ss DNA as if it were apart of a ds duplex
26
what is the function of the RecA proteins?
- The RecBCD complex processes the 3’ end making it suitable for RecA to facilitate formation of a triple stranded helix
- Assimilation of invading strands
- Aggregates into long filaments with ss or ds DNA: 6 RecA monomers per turn of filament which has a deep groove containing the DNA and 3 nucleotides per RecA monomer
- each RecA monomer present a nucleotide triplet to the dsDNA to quickly scan for homology; at sites of homology, strand exchange occurs between ssDNA and the homologous strand in the dsDNA
- DNA held in form that is 1.5 times extended from normal B form (one turn per 18. nt)
- First step is for RecA to bind to ssDNA then dsDNA
- Interaction of the two DNAs takes place in the filament groove before actual strand exchange (which requires a 3’ nick) ss DNA is bound first. At the end of the reaction, RecA is bound to dsDNA
- Large amounts of ATP are hydrolyzed during the release of recA from the duplex DNA. ATP may act in an allosteric fashion, changing the affinity of RecA for DNA: high affinity when ATP is present, low affinity when absent
- Initial heteroduplex DNA may not be plectonemic relationship in a paranemic joint the two strand lie side by side without intertwining
- ssDNA is spread out in a conformation similar to its conformation in ds DNA
- helical ssDNA with RecA can pair with dsDNA in its major groove to form a triple-stranded helix
- the strands of the invaded dsDNA are displaced and can then pair with respective strands of the invading DNA, D-loop and holliday junction forms
27
what are the homologs to RecA?
- human Dmc1
- rAD51
28
How is the triple stranded helix formed?
- RecBCD hands off to RecA for strand invasion
- Scans duplex DNA
- Ss that is coated with RecA is doing the invading
- RecA has a primary binding site for ssNDA and a secondary transient binding site for dsDNA
- Each RecA monomer presents a nucleotide triplet to the dsDNA to quickly scan for homology
- At sites of homology, strand exchange occurs between the ssDNA and the hoologos strand in the dsDNA
- Helical ssDNA with RecA can pair with dsDNA in its major groove to form a triple stranded helix
- The strands of the invased dsDNA are displaced and can then pair with respective strands of the invading DNA - displacement loop - holliday junction
29
paranemic helix in vitro
- linkage is easily destroyed by incubating for 5 minutes at 20 degrees below the melting temperature
- very unstable, 50 bo
- nicking not required to initiate synapsis in vitro
30
branch migration
expands the displacement loop to facilitate 5' end capture. the site of the original strand invasion may be hundreds of bases away
31
Ruv A
recognizes the structure of the junction
32
Ruv B
a helicase that catalyzes branch migration
- a heximeric helicase that binds double-strand DNA upstream of the crossover point
33
Ruv C
cleaves junctions to generate recombination intermediates
- an endonuclease that resolves the holliday junction
- recognizes the sequence 5' ATTG 3' for cutting
- it is thought that the orientation of this sequence may influence the choice of strands
34
RuvABC system
- moves at 10-20 bp/sec
- RUVAB displaces RecA during branch migration
35
RecG
another helicase that can substitute for RuvB
- if both genes are mutated, recombination is blocked
36
what are the similar complexes mammals have to the RuvABC system that resolves holliday junctions?
- GEN1 and MUS81
37
what is the function of the RuvABC system?
- After RecA initiates strand exchange, branch migration is conducted by RuvAB
- Displaces RecA during branch migration
- RuvC is the endonuclease that resolves the Holliday junction, occurs at tetramer site that may direct direction of cuts
- Migration increases the length of heteroduplexes, allowing for isomerization of holliday junctions
- RuvA binds and stabilizes holliday junction
- RuvB then binds to RuvA/DNA complex
- RuvC binds resolves holliday junction by cleaving the two crossed strands
