Lecture 7 - Homologous Recombination

Front 1

Five things homologous DNA recombination used for?

Back 1

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

Front 2

2 ways DNA damage leads to double strand breaks during DNA replication
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Back 2

A nick or lesion in template strand

Front 3

A nick in template strand leads to...

Back 3

fork collapse and double strand blunt end needed for homologous recombination. The broken end can serve to initiate recombination.

Front 4

A lesion in the DNA template strand leads to...

Back 4

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.

Front 5

Two models of homologous recombination...

Back 5

1) Holliday and 2) Double stranded break model

Front 6

Double-stranded break model also known as...

Back 6

RecBCD model

Front 7

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

Back 7

heteroduplex DNA

Front 8

Holliday model for DNA recombination:
junction resolution. What are the two outcomes depending on the cut?

Back 8

1. Splice or 2. Patch

Front 9

Splice is a ____________ cut and produces __________ products.

Patch is a ____________ cut and produces ____________ products.

Back 9

vertical, crossover

horizontal, noncrossover

Front 10

Explain the DSB break model

Back 10

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

Front 11

No enzyme in E. coli has been found to make...
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Back 11

double strand breaks

Front 12

so recombination is initiated through...
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Back 12

DNA damage, stalled replication forks, or introduction of foreign linear double stranded DNA into the cell

Front 13

A complex of _____, ______, ______, binds the double-stranded end of DNA and proceeds down the DNA unwinding it and degrading both strands
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Back 13

RecB, RecC, and RecD (RecBCD)

Front 14

Once a _____________ site (GCTGGTGG) in DNA is encountered, only one strand (possessing the __ end of DNA) of the two is now degraded.

Back 14

chi (crossover hot spot instigator), 5’

Front 15

This then forms a single stranded DNA molecule with the ____ site at the ____ end.
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Back 15

chi, 3'

Front 16

This single stranded DNA is covered by the _____ protein. binds every __ nucleotides of DNA.
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Back 16

RecA, 3

Front 17

RecA forms _________ in a ________ direction on single stranded DNA
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Back 17

filaments, 5’-> 3’

Front 18

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.

Back 18

sequence complementarity, strand displacement

Front 19

_____ 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)

Back 19

RuvA, RuvB, RuvC

Front 20

In eukaryotes _____ protein
forms double strand breaks
to initiate recombination
in _______

Back 20

Spo11, meiosis

Front 21

_________ residue on spo11 binds phosphate group
On ____ end.

Back 21

tyrosine, 5'

Front 22

In humans we need to initiate dbl stranded break to
induce recomb. It isnt a ______ even. Break isnt _______.
About a 2bp overhang.

Back 22

random, blunt

Front 23

____ complex in eukaryotes
functions as RecBCD in E. Coli
to create a single stranded region
of DNA.

Back 23

MRX

Front 24

Formation of the DSBs during meiosis requires the presence of both _____ and the ______ complex

Back 24

Spo11 and MRX

Front 25

_____ protein is responsible for 5' --> 3' resection at the break site.

Back 25

MRX

Front 26

The strand-exchange proteins _____ and _____ then assemble on the ssDNA tails.

Back 26

Dmc1, Rad51

Front 27

Dmc1 and Rad51 are _____
homologs that bind the single
stranded DNA and stimulate
strand invasion at __________________________.

Back 27

RecA, homologous
sequences

Front 28

________________ protein resolves
Holliday junctions similar to
________ in E. Coli

Back 28

Rad51c-XRCC3, RuvC

Front 29

Recombination steps in E.coli:

Pairing homologous DNA and strand invasion

Back 29

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.

Front 30

Introduction of DSB

Back 30

none

Front 31

Processing DNA breaks to generate ss for invasion

Back 31

RecBCD helicase/nuclease

slide 13 of lecture 7

Front 32

Holiday junction recognition and branch migration

Back 32

Ruv AB complex


RuvA tetramer protein binds the Holliday junction. RuvA recruits the
hexameric ATPase RuvB which provides the energy for branch
migration

Front 33

Resolution of holiday junctions

Back 33

RuvC

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