L8: Homologous Recombination Flashcards
define recombination
physical exchange of DNA between chromosomes
what does homologous recombination need?
- double stranded breaks (DSBs) in DNA
- homology
what does homologous recombination need? - double stranded breaks in DNA
- repaired by 2 mechanisms:
1. non-homologous end-joining (NHEJ)
2. homologous recombination
what does homologous recombination need? - homology
- two DNA sequences are identical or nearly identical for a substantial distance
- small variations can exist due to presence of variable alleles
what does homologous recombination need?: homology - define variable alleles
different versions of the same genetic locus
explain the steps of the DSB-repair pathway
- DBS occurs in only one of two homologous DNA molecules
- the break is enzymatically processed to give 3’ ssDNA extensions
- ssDNA extensions invade intact homologous DNA and anneal to complementary regions
- 3’ termini primes DNA synthesis using the homologous strand as template
- branches migrate and two holliday junctions form
holliday structure resolution
- can be cleaved in two planes and can be resolved in two ways:
- spliced or patched method
spliced or crossover product
- cleavage through strands composed entirely of parental DNA
- genes flanking recombination sites are re-assorted
patched or noncrossover product
- cleavage through strands composed of both types of parental DNA
- genes flanking recombination sites are not re-assorted
what is RecA
- its involved in the homologous recombination in prokaryotes
- it is a strand exchange protein that facilitates recombination
RecA - how does it facilitate recombination?
- binds ssDNA to form a filament
- looks for complementarity with double stranded DNA (dsDNA) molecules
- dsDNA transiently opened and tested for complementarity with ssDNA
RecA - where is ssDNA and dsDNA located?
- ssDNA: primary binding site
- dsDNA: secondary binging site
what is gene conversion
- its happens due to patch/noncrossover event
- can replace a gene with a different allele
gene conversion - explain mate-type switching in S. cerevisiae
- haploid cells are of two mating types: a and alpha
- cells of different mating types can fuse to form a diploid organism
- mating-type genes encode transcriptional regulators
gene conversion - transcriptional regulators
- found at the mating-type (MAT) locus:
1. MATa locus (gene a)
2. MATalpha locus (genes alpha 1, alpha 2)
how can recombination happen with mate-type switching in S. cerevisiae
- both mating types (a and alpha) have silent copies of a and alpha genes, HML and HMR
- an OH endonuclease cleaves recognition sites at AT locus to generate DSB
- the stand invades HML (if MATa) or HMR (if MATalpha)
- DSB is repaired via gene conversion resulting in recombination
- recombination is unidirectional with only the MAT locus changing
mate-type switching - what is the mechanism behind this recombination
- not Holliday structure bc it doesn’t generate crossover products
- it is instead synthesis-dependent strand annealing (SDSA)
what is the SDSA model?
- HO endonuclease cleaves MAT locus
- the invading 3’ ssDNA strand serves as primer to initiate DNA synthesis
- simply uses silent locus as template to synthesize new gene
- other 3’ ssDNA strand is cleaved by an endonuclease
- newly synthesized strand serves as template to copy the new allele
