L7 - Machinery of Homologous recombination

Question 1

mechanism of homologous recombination with enzymes

Answer 1

1. RecBCD causes end resection to create 3’ overhangs

= unwinds DNA and cleaves

2. RecA coats 3’ end and seaches undamaged DNA for complimentary DNA

= homology search and strand invasion

3. RuvA binds to holiday junction formation acting as a platform
4. RuvB binds and causes branch migration

= RuvB is a helicase

5. RuvC cleaves holiday junctions in resolution

Question 2

what does RecBCD recognise on 3’ end of DNA that means its nit digested as much as 5’ - end resection

Answer 2

Chi sequence

= conformational change means it doesnt digest 3’ as much

Question 3

which domain of RecBCD is a nuclease compared to the other 2 which are helices

Answer 3

RecC

Question 4

why do the 3’ ends need coating by RecA

Answer 4

protection from degredation

= ssDNA is uncommon and targetted for destruction

Question 5

role of RuvA

Answer 5

binds to holiday junction

forms a tetramer = 4 monomers

= acts a s aplatform fro other porteins to be recruited

Question 6

role of RuvB

Answer 6

motor protein/helicase

drags DNA through RuvAB complex

causes branch point migration = no nicks in dsDNA

Question 7

role of RuvC

Answer 7

Endonuclease

dimer protein that has a preffered sequnce for cleavage

= ensures the same cut is done on each holiday junction

= where it cuts affects the outcome products (crossover/non-crossover)

Question 8

how does RecA work in homology search and strand invasion

Answer 8

forms filaments with other recAlls monomers

binds with high affinity to ssDNA and low affinity to dsDNA

= samples dsDNA randomly 3bp at a time
= tests for complimentarity against the ssDNA

Question 9

what does recA do to dsDNA to test it for complementarity

Answer 9

stretches dsDNA bound in the low affinity binding site

= unstable base paires popout of sequnce
= if complimntary = H bonds to Base on ssDNA

Question 10

homologous recombination in restarting the replication fork

Answer 10

replication forks can break down when trying to replicate pask a nick

= produces 2 ds DNA molecules - 1 short + 1 long

1. end proscessing creates 3’ overhang on short DNA
2. invades and is extended
3. Nick is introduced on the other strand by endonuclease
4. branch migration resolves the SINGULAR holiday junction
5. nick is healed

= replication fork can restart

Question 11

crossing over/ homologousb recombination in meiosis

Answer 11

programmed/deliberate HR produces new combinations of maternal and paternal chromosones

= via corssover producst so material is exchanged between the chromosones

Question 12

crossover products vs gene conversion in meiosis due to deliberate homologous recombination

Answer 12

crossover:
exchanhing maternal and paternal alleles between chromosomes to create new combinations

gene conversion:
non-reciptocal exchange of genes between chromosomes - taken a way a gene and replaced by a similar but not identical gene

= due to mismatch of base pairs during strand invasion

Question 13

what is a possible result of gene conversion

Answer 13

Loss of heterozygosity

loss of cerain types of alleles or both paternal and maternal having the same - homozygous

Question 14

what is the name of the nuclease enzyme that causes deliberate ds breaks in DNA

Answer 14

Spo11

Question 15

what is Spo11

Answer 15

causes puposefulds breaks in DNA

not seuqnce specific BUT there are ‘hotspots’ for where DNA is more likely to be cut

2 Spo11s needed = 1 per strand