Recombination Flashcards
what are the 2 purposes of homologous recombination
1) evolution, the increase in genetic variation
a) changing gene order
b) moving genes to different replicons (transformation/transduction/conjugation/transposition)
2) facilitation of DNA repair to save from cell death
- restart of DNA replication
what is site specific recombination
requires special proteins that promote recombination in specific sequences
what is homologous recombination
occurs often
- recombination between any 2 similar DNA sequences
who discovered DNA recombination and how was it discovered
discovered by hershey and rotman
- coinfected e-coli with 2 different but related DNA and found progeny had 98% parental genes but also 2% of genes that were unlike parental.
- came up with copy choice and breakage & reunion model of recombination
what is the copy choice model of recomb
that proteins copy different templates if DNA and bring them together in homology to form recombinant
what is breakage and reunion model of recomb
that proteins cut and shuffle DNA pieces and ligase them together
what did meselson and weigles experiment reveal
they coinfected e-coli with a heavy and light lamda DNA
- recombinant progeny density was examined. if density changes then it favors breakage and reunion model of recomb.
their conclusions suggest that since there was change in density, recombination involves physical exchange of genetic material
what are the requirements for homologous recombination
1) similar sequences in the cross over region
2) complementary base pairing for synapse between 2 dsDNA
3) recombination enzymes (nucleases/ligases)
4) formation of heteroduplexes
what is synapse
the point where 2 DNA molecules are complementary paired for crossing over
what is the holliday model of recombination
1) 2 dsDNA undergo single stand nick at identical sites
2) single strands cross over (stand invasion) to create 2 heteroduplexes (holiday junction)
3) branch migration (moving of holiday junction) influences length of genetic material being exchanged/ crossed over
4) holliday junction is cleaved either horizontally (patch product) or vertically (flanking product) by resolvase
what is patch product of holliday resolution
contains regions of non crossing over, parenting strands still intact
from horizontal cleavage
what is flanking product of holliday resolution
contains regions of crossing over between 2 different strands, from vertical cleavage
what is single strand invasion model of recombination
1) one strand of one of the dsDNA is cut and invades another dsDNA until it finds complementary sequence
2) displaces segment of dsDNA strand and degrades as it recombines with its other strand.
meanwhile DNA pol fills in gap left by invading strand creates holliday junction.
3) branch migration and resolution occurs
what is double stranded break and repair model of recombination
1) both strands of 1 DNA molecule is broken
2) 5’ ends digested to reveal 3’ overhands
3) single strand invades other dsDNA strand and displaces strand to create d-loop
4) polymerization via dna pol fills the gaps. invader ssDNA crosses over again to return to previous DNA duplex= 2 holliday junctions
5) cleavage and resolution
what is the difference between DNA replication and DNA recombination for DNA repair
1) initiation of DNA replication requires protein synthesis with no recombination functions
2) recombination uses stable DNA replication which does not require protein synthesis
summarize the process of homologous recombination
1) single stranded DNA end is generated through RecBCD
2) recA coats single ssDNA and looks for complementary strands to form synapse and holliday junction
3) RuvABC and RecG catalyze resolution of holliday junction
what are the general functions of RecBCD
- have helicase properties which unwind dsDNA (recBD)
- have endonuclease properties which cleave/degrade DNA (recB exonuclease)
- has DNA ATPase for energy generation
what is the chi site (and the process to it)
RecD helicase unwinds DNA as recB exonuclease cleaves the strands and they degrade
chi site is a sequence that signals RecC to inhibit recD activity, this activates RecB helicase and now 3’-5’ end remains uncleaved where recA will bind for subsequent processes
what is the recBCD pathway to molecular recombination
1) double strand break occurs in single DNA molecules
2) RecD helicase unwinds DNA as recB exonuclease cleaves the strands and they degrade
3) @ chi sequence, RecC inhibits recD activity, this activates RecB helicase and now 3’-5’ end remains uncleaved
4) recA binds to 3’-5’ stand to look for complementary strand in other duplex through strand invasion (D-loop)
5) DNA pol fills gaps, ligase glues= 2 holliday junctions
6) branch migration occurs with ruvAB and ruvC resolves
what are the roles of Rec BCD on recA
RecBC–> loads recA where recombination would occur
recD–> inhibits recA binding before chi site
recA–> binds in helical structure
what are the roles of chi sites
1) used as defense against foreign or potentially toxic DNAs
any DNA without chi site is degraded before recomb
2) controls orientation of DNA break so that recombination repair occurs in same direction as Ori C
what is the recF pathway
occurs when there is a stalled lesion on DNA
1) recQ helicase unwinds DNA with recJ exonuclease
2) recFOR recruits recA which follows standard recomb after
what is transactivation
process by which recA can activate a non complementary dsDNA to allow invasion of complementary ssDNA to form d-loop and look for complements on strand
what is spooling
ability by which recA brings in DNA and matches it up throughout in prep for homologous recomb.
