Unit 16 - Homologous Recombination

What is the definition of recombination?

a process by which pieces of DNA are broken and recombined to produce new combinations of alleles

What does recombination between homologous DNA sequences provide?

genetic variation, mechanism to repair DNA, restarting of stalled replication forks

What are two different ways that double-stranded breaks are formed?

nick in template strand and DNA lesion in template strand

What forms the nick in template strand?

ionizing radiation

What forms the DNA lesion in template strand?

thymine dimers

Explain how a nick in the template strand causes DSB

nick will go through one DNA strand, and the fork will collapse forming a break in one of the produced DNA strands

Explain how a DNA lesion forms a DSB

as replication encounters a lesion, the fork collapses and replication is arrested, there is fork regression producing a DSB

What are the key steps in homologous recombination

alignment of two homologous DNA molecules, introduction of breaks in the DNA, strand inversion, holliday junction formation, and resolution of the holliday junction

What is the 5' to 3' resection?

where the 5′ ends of DNA at a double-strand break (DSB) are chewed back, or resected, to leave behind 3′ single-stranded overhangs

What does the 5' to 3' resection provide?

3' overhang that can invade the double stranded DNA

What is the holliday junction?

cross-shaped structure that forms during the process of genetic recombination, when two double-stranded DNA molecules become separated into four strands in order to exchange segments of genetic information.

What happens when the holliday junction is cleaved vertically?

crossover occurs, where there is reassortment of flanking genes

What happens when the holliday junctions are cleaved horizontally?

noncrossover, results in a patch and no recombination of genes

true or false: the process of DSBR occurs twice because there is two sides of DNA that are broken

true

true or false: recombination always results in crossover of genes

false

How does the double holliday junctions result in crossover?

cleaving at site 1 at junction x and cleaving at site 2 at function y

How does the double holliday junction result in noncrossover?

cleaving at site 2 (at holliday junction) at both junction x and y

true or false: homologous recombination is mediated by specialized proteins that are regulated for this process

true

What is the RecBCD helicase/nuclease utilized for?

processing DNA breaks to generate single strands for invasion

What is the function of RecB in the RecBCD complex?

3' to 5' helicase + nuclease

What is the function of RecD in the RecBCD complex?

5' to 3' complex

What is the function of RecC in the RecBCD complex?

recognizes the chi site

Explain how the RecBCD is utilized in E. coli

RecBCD enters at DSB and RecD reads fast while RecB reads slow, RecC recognizes the chi site which will pause degradation, the RecD will continue the degradation while RecB does not degrade any farther

true of false: RecBCD requires ATP to work

true

What enzyme performs the 5' to 3' resection?

RecBCD in e. coli

What is the polar action of the chi site?

the closer sequences are to the chi site the higher the probability of recombination

true or false: if e. coli DNA enters an E. coli cell it will most likely recombine

true

What protein helps to pair homologous DNAs and strand inversion?

RecA proteins in E. coli and Rad51 in eukaryotes

Describe the RecA proteins

strand-exchange protein which promote search for sequence matches and generate base pairing between them

What is the active form of the RecA enzyme?

RecA filaments

What does the RecA filaments do to the DNA?

extends distance between the two adjacent nucleotides

true or false: RecA-like filaments are not conserved throughout life forms

false

What is the polarity of the RecA filaments?

will only bind in the 5' to 3' direction of the ssDNA

How does the RecA filament bind?

cooperatively

What will like the RecA filaments not bind to?

molecules with 5' ssDNA extensions, can only bind to 3' overhangs

Explain the strand-exchange reaction

RecA protein ssDNA displaces one of the strands that is not complimentary

What proteins facilitates the holliday junction recognition and branch migration?

RuvAB complex

How does the RuvAB complex promote branch migration in bacteria?

exchange of DNA base pairs between duplexes

What is the function of RuvA?

binds to holliday junctions and recruits RuvB

What is the function of RuvB

ATPase which is similar to hexameric helicase involved in replication

What protein is utilized in the resolution of holliday junctions?

RuvC resolvase

What does the RuvC do?

nicks the two strands with the same polarity

What sequence does the RuvC cleave?

5'-A/T-T-TG/C-3'

What is required for chromosome segregation during meisosis?

meiotic recombination

true or false: introduction of dsb only occurs in eukaryotes

true

What is the meiotic crossovers initiated by DSBs initiated by?

Spo11 endonuclease

How does the Spo11 endonuclease work?

Spo11 contains hydroxyl groups and contains tyrosine site attacks to the phosphodiester backbones

Where do the Spo11 target?

two subunits, two nucleotides apart

Describe the meiotic recombination pathway

utilizes Spo11, MRX complex, Dmc1, and Rad51 to recombine the chromosomes

What proteins are included in the MRX complex?

Mre 11, Rad50, and Xrs2

What is the MRX complex do?

performs the resection, equivalent to RecBCD

Rad51 and DMC1 are used to...

coat the overhangs and promote the strand-exchange

What is the difference between Rad51 and DMC1?

DMC1 is meiotic cell-specific and preferentially between non-sister chromatids

What is a recombination factory?

large protein-DNA complexes that participate in recombination process

true or false: the Rad51 interacts with tumor supressor gene BRCA2 in repairing DSBs

true

Why does mutations in BRCA2 cause half of familial type of breast cancer?

a mutation in the Rad51 would cause there to be no homologous end-joining, further increased mutations