11. Homologous Recombination

Recombination

exchange of genetic material between two DNA molecules: causes DNA rearrangement

biological Roles of REcombination

1. DNA repair

2. creation of new gene/allele combinations (through corssing over during meiosis)

3. formation of new genes (IG rearrangement)

4. integration of a specific DNA element

Experimental Uses of Recombination

1. gene mapping (distance between genes estimated through recombination frequency)

2. making transgenic cells and organisms

general or homologous recombination

genetic exchange between pair of homologous DNA sequences

Site specific recombination

occurs between sequences with a limited stretch of similarity: involves specific sites (recombination sites)

Transposition

mobile DNA element moves from one site to another (donor and target site), usually little sequence similarity involved

genetic exchange exchange in homologous DNA sequences can occur

1. between two copies of the same chromosome = sister chromatids (mostly in DNA repair in both prokaryotes and eukaryotes

2. bewteen homoloous chromosomes = crossing-over in meiosis; also in a type of DNA repair in eukaryotes

Key steps in SS break model

1. ALIGNMENT of two homologous DNA molecules

2. INTRO of the break in one strand of each homologous DNA

3. STRAND INVASION: ss region from one of the parental strand’s pairs with the complementary strand from the homologous DNA molecule

Outcome of SS break

two molecules become connected through crossing DNA strands

cross structure

Holiday junction

branch migration

movement of holliday junction

Cleavage of holliday junction

resolution

“splice” or crossover products - recombination between A and C

ABC → ABc and abc → abC

patch or no cross over products - no reassortment

ABC → AbC and abc → aBc

Model for homologous recombination -DS break

Similar steps in prokaryotic and eukaryotic DSB replication repair and eukaryotic meiosis; different recombination proteins involved

Szostak model

1. double strand break in one of the homologous dsDNA

2. exonucleases extend the gap, producing 3’ overhangs

3. the exposed 3’ end base pairs with its complement in the intact gomolog (rec proteins)

4. extension at 3’ end via DNA polymerase and branch- migration and formation of 2nd crossover creating two holliday junctions

   CELL MUST RESOLVE HOLLIDAY JUNCTIONS

5. cleavage of holliday junctions

in DS break, if resolution only occurs at site 2 in both x and y

produces non-crossover products

in DS break, how to produce crossover products

resolution of x at site 1 and y at site 2

bacteria (e.coli has only one chromosome) its haploid, major role of homologous recombination

in bacteria is to repair DSB

note: recombination (not necessarily homologous) can occur between bacterial chromosome and “shorter incoming DNA thorugh”

1. transformation

2. transduction

3. conjugation

transformation

cell can absorb and integrate fragments of DNA from their environment

Transduction

viruses transfer genes between prokaryotes

conjugation

one cell directly transfers genes (carried by e.g plasmid) to another cell

Chi - Crossover Hotspot instigator

• short DNA sequence that acts as a hotspot for recombination

• role is to increase the frequency of homologous recombination

RecBCD

nuclease/helicase; binds to duplex DNA and generates single srands for invasion - REQUIRE ATP

RecA

starts recombination by facilitating pairing of homologous DNAs: involved in strand invasion

RuvAB

complex with helicase activity, recognizes holliday junctions, catalyses branch migration

RuvC

endonuclease that catalyses the resolution of Holliday junction

RecBCD/Chi mechanism

1. RecBCD complex enters teh DNA at the site of the double strand break

2. unwinds and degrades both strands of DNA int he presence of ATP

3. upon reaching a chi sequence, activity changes - chi sequence controls RecBCD activity (stilll unknown how)

4. degradation of 3’ end stops, degradation of 5’ end increases creating 3’ overhang

5. RecA binds to the overhand and initiates strand exchange

6. branch migration by RuvAB and holliday junction resolution by RuvC

RecBCD destroys any

foreign DNA - which typically lacks chi sequences

programmed generation of DSBs occurs during

meiosis

homologous recombination required for proper

chromosome pairing during meiosis (otherwise chromosomes fail align)

• gene reshuffling - variability !

SPO 11

may be Topo II-like enzyme as it generates double strand breaks

Mre 11 is

nuclease (5’-3’ digestion; leaves 3’ overhangs)

Dmc1 and Rad1 are

RecA type strand-exchange (strand invasion) proteins

mismatch repair system recognizes mis-paired bases in heteroduplex (x/x)

• it excises and replaces one of the strands to restore complentarity

• conversion of one allele into another

• transfer of genetic information is nonreciprocal gene conversion

• source of non-Mendelian inheritance patterns

gene conversion

• nonreciprocal transfer of genetic information from one DNA molecule (or allele0 to another

• instead of both DNA molecules exchanging sequences equally (as in a crossover) one allele’s sequence is replaced with others