Recombination

Question 1

what are the 2 purposes of homologous recombination

Answer 1

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

Question 2

what is site specific recombination

Answer 2

requires special proteins that promote recombination in specific sequences

Question 3

what is homologous recombination

Answer 3

occurs often
- recombination between any 2 similar DNA sequences

Question 4

who discovered DNA recombination and how was it discovered

Answer 4

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

Question 5

what is the copy choice model of recomb

Answer 5

that proteins copy different templates if DNA and bring them together in homology to form recombinant

Question 6

what is breakage and reunion model of recomb

Answer 6

that proteins cut and shuffle DNA pieces and ligase them together

Question 7

what did meselson and weigles experiment reveal

Answer 7

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

Question 8

what are the requirements for homologous recombination

Answer 8

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

Question 9

what is synapse

Answer 9

the point where 2 DNA molecules are complementary paired for crossing over

Question 10

what is the holliday model of recombination

Answer 10

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

Question 11

what is patch product of holliday resolution

Answer 11

contains regions of non crossing over, parenting strands still intact
from horizontal cleavage

Question 12

what is flanking product of holliday resolution

Answer 12

contains regions of crossing over between 2 different strands, from vertical cleavage

Question 13

what is single strand invasion model of recombination

Answer 13

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

Question 14

what is double stranded break and repair model of recombination

Answer 14

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

Question 15

what is the difference between DNA replication and DNA recombination for DNA repair

Answer 15

1) initiation of DNA replication requires protein synthesis with no recombination functions

2) recombination uses stable DNA replication which does not require protein synthesis

Question 16

summarize the process of homologous recombination

Answer 16

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

Question 17

what are the general functions of RecBCD

Answer 17

• have helicase properties which unwind dsDNA (recBD)
• have endonuclease properties which cleave/degrade DNA (recB exonuclease)
• has DNA ATPase for energy generation

Question 18

what is the chi site (and the process to it)

Answer 18

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

Question 19

what is the recBCD pathway to molecular recombination

Answer 19

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

Question 20

what are the roles of Rec BCD on recA

Answer 20

RecBC–> loads recA where recombination would occur
recD–> inhibits recA binding before chi site
recA–> binds in helical structure

Question 21

what are the roles of chi sites

Answer 21

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

Question 22

what is the recF pathway

Answer 22

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

Question 23

what is transactivation

Answer 23

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

Question 24

what is spooling

Answer 24

ability by which recA brings in DNA and matches it up throughout in prep for homologous recomb.

Question 25

which proteins mediate isomerization and resolving of holliday junctions

Answer 25

1) ruvA–> structure by which holliday junction becomes planar
2) ruvB–> structure that uses ATP to pump DNA through the junction in search for cleavage sequences
3) RuvC–> structure that cuts at 2 specific ssDNA sites

Question 26

where do linear DNA come from for recombination

Answer 26

conjugation
transduction (they can encode for recomb proteins similar to e-coli)
transformation

Question 27

what is recA

Answer 27

proteins that enhance pairing of homologous DNA and synapse formation

Question 28

what is recBC

Answer 28

has helicases, exonucleases, and chi-specific endonuclease, ATPase

initiates recombination through helicase activity, degrades DNA until chi site, and loads recA

Question 29

what is recD

Answer 29

stimulates exonucleases by degrading 3’ ends, inhibited by recC at chi site

Question 30

what is ruvA

Answer 30

protein that binds to holliday junctions and recruits ruvB
also migrates holliday junctions (branch migration)

Question 31

what is ruvB

Answer 31

holliday junction specific helicase involved in branch migration of junction

Question 32

what is ruvC

Answer 32

holliday junction specific nuclease which resolves junction