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33 Cards in this Set

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Five things homologous DNA recombination used for?
1. Used for repair of DNA, especially for repairing double strand breaks

2. Can be used to restart stalled replication forks

3. Used in meiosis in germ cells for pairing of chromosomes and crossing over

4. Used to incorporate DNA that has entered E. coli by conjugation or phage infection

5. Used to degrade foreign double stranded DNA in E. Coli
2 ways DNA damage leads to double strand breaks during DNA replication
A nick or lesion in template strand
A nick in template strand leads to...
fork collapse and double strand blunt end needed for homologous recombination. The broken end can serve to initiate recombination.
A lesion in the DNA template strand leads to...
fork collapse and fork regression.

In this case, the two newly synthesized strands (red) can basepair and the fork can regress. This structure can be further processed by a number of means. The broken end can serve to initiate recombination.
Two models of homologous recombination...
1) Holliday and 2) Double stranded break model
Double-stranded break model also known as...
RecBCD model
As a result of the strand invasion process, regions of new duplex DNA are generated; this DNA, which often contains some mismatched base pairs, is called
heteroduplex DNA
Holliday model for DNA recombination:
junction resolution. What are the two outcomes depending on the cut?
1. Splice or 2. Patch
Splice is a ____________ cut and produces __________ products.

Patch is a ____________ cut and produces ____________ products.
vertical, crossover

horizontal, noncrossover
Explain the DSB break model
1. DSB in one of two homologous duplex DNA molecules
2. Processing to generate gapped DNA with 3' ss tails
3. Strand invasion of 3' end to other DS DNA
4. Second strand invasion and DNA repair synthesis from 3' ends
5.Branch migration and formation of an intermediate with two holliday junctions
No enzyme in E. coli has been found to make...
double strand breaks
so recombination is initiated through...
DNA damage, stalled replication forks, or introduction of foreign linear double stranded DNA into the cell
A complex of _____, ______, ______, binds the double-stranded end of DNA and proceeds down the DNA unwinding it and degrading both strands
RecB, RecC, and RecD (RecBCD)
Once a _____________ site (GCTGGTGG) in DNA is encountered, only one strand (possessing the __ end of DNA) of the two is now degraded.
chi (crossover hot spot instigator), 5’
This then forms a single stranded DNA molecule with the ____ site at the ____ end.
chi, 3'
This single stranded DNA is covered by the _____ protein. binds every __ nucleotides of DNA.
RecA, 3
RecA forms _________ in a ________ direction on single stranded DNA
filaments, 5’-> 3’
RecA-ssDNA then scans double stranded DNA looking for _____________________ (does not need to be identical).

Once such sequence is found, _________________ for several hundred base pairs occurs leading to Holiday junction formation.
sequence complementarity, strand displacement
_____ tetramer protein binds the Holliday junction. it recruits the
hexameric ATPase ____which provides the energy for branch
migration. _____ endonuclease cleaves 2 strands at each
junction to finish recombination. RuvC cleaves at A/T-T-T-G/C sites. (4 base sequence with two T’s in the middle)
RuvA, RuvB, RuvC
In eukaryotes _____ protein
forms double strand breaks
to initiate recombination
in _______
Spo11, meiosis
_________ residue on spo11 binds phosphate group
On ____ end.
tyrosine, 5'
In humans we need to initiate dbl stranded break to
induce recomb. It isnt a ______ even. Break isnt _______.
About a 2bp overhang.
random, blunt
____ complex in eukaryotes
functions as RecBCD in E. Coli
to create a single stranded region
of DNA.
MRX
Formation of the DSBs during meiosis requires the presence of both _____ and the ______ complex
Spo11 and MRX
_____ protein is responsible for 5' --> 3' resection at the break site.
MRX
The strand-exchange proteins _____ and _____ then assemble on the ssDNA tails.
Dmc1, Rad51
Dmc1 and Rad51 are _____
homologs that bind the single
stranded DNA and stimulate
strand invasion at __________________________.
RecA, homologous
sequences
________________ protein resolves
Holliday junctions similar to
________ in E. Coli
Rad51c-XRCC3, RuvC
Recombination steps in E.coli:

Pairing homologous DNA and strand invasion
RecA protein

RecA-ssDNA then scans double stranded DNA looking for
sequence complementarity (does not need to be identical). Once such sequence is found, strand displacement for several hundred base pairs occurs leading to Holiday junction formation.
Introduction of DSB
none
Processing DNA breaks to generate ss for invasion
RecBCD helicase/nuclease

slide 13 of lecture 7
Holiday junction recognition and branch migration
Ruv AB complex


RuvA tetramer protein binds the Holliday junction. RuvA recruits the
hexameric ATPase RuvB which provides the energy for branch
migration
Resolution of holiday junctions
RuvC

RuvC endonuclease cleaves 2 strands at each
junction to finish recombination. RuvC cleaves at A/T-T-T-G/C sites