Chiolo Lecture 7

Question 1

What initiates resection?

Answer 1

1. MRN-CtIP endonuclease
2. ATP hydrolysis

Question 2

What extends resection?

Answer 2

Exo1 exonuclease and Dna2 endonucleases

Question 3

What binds to ssDNA following resection separation?

Answer 3

RPA

Question 4

What is the MRN complex organized into?

Answer 4

different conformations for HR and NHEJ

Question 5

What components makeup the MRN complex?

Answer 5

1. Mre11
2. Rad50
3. Nbs1

Question 6

What does the MRN-CtIP complex generate?

Answer 6

a nick downstream from the DSB site to initiate resection

Question 7

What is the function of RPA?

Answer 7

single stranded binding protein that keeps DNA unwound for DNA replication/repair

Question 8

What is cooperative binding?

Answer 8

the binding of one protein facilitates the next; RPA causes the binding of additional RPA

Question 9

What is the function of RecA/ Rad51?

Answer 9

1. binds to ssDNA to drive strand invasion
2. RecA/ Rad51 forms a filament around the ssDNA and probes for homology

Question 10

How does RecA/Rad51 bind to ssDNA?

Answer 10

substitutes RPA

Question 11

How many base pairs are required to send the signal for continued pairing?

Answer 11

15 base pairs

Question 12

What is the process of RecA/ Rad51 homology?

Answer 12

1. ssDNA binds to the primary DNA binding site
2. dsDNA binds to the secondary site to test for complementarity
3. base pairs are switched

Question 13

What is requried for Rad51 recruitment in mammalian cells?

Answer 13

Brca2

Question 14

What is the function of Brca2?

Answer 14

substitutes RPA with Rad51

Question 15

What is function of OB-fold domain?

Answer 15

binds and loads 6 Rad51 molecules at a time

Question 16

What is a result of Brca2 mutation?

Answer 16

breast cancer

Question 17

What are the risks of breast cancer?

Answer 17

1 in 8 women

Question 18

What is needed for resection extension?

Answer 18

DNA2 endonuclease + Blm or Wrn 3’-5’ helicase

Question 19

What is a result of Wrn mutation?

Answer 19

Werner syndrome

Question 20

What are the symptoms of Werner syndrome?

Answer 20

1. cancer predisposition
2. telomeric instability
3. premature aging

Question 21

What is the function of Blm?

Answer 21

Blm helicase reverts recombination intermediates by displacing Rad51; this is useful in case of strand invasion of the wrong sequence - prevents aberrant recombination

Question 22

What results from Blm mutations?

Answer 22

Blood syndrome

Question 23

What are the symptoms of Blood syndrome?

Answer 23

1. cancer predisposition
2. UV sensitivity
3. sterility
4. chromosome abnormalities

Question 24

What is the function of RuvAB complex?

Answer 24

promotes Holliday junction migration in bacteria

Question 25

What is the function of RuvA?

Answer 25

recognizes Holliday junctions

Question 26

What is the function of RuvB?

Answer 26

promotes movement (uses ATP)

Question 27

What is the function of RuvC?

Answer 27

1. resolves Holliday Junctions by cleaving across the Junction 2. recognizes Hjs in complex with RuvAB 3. introduces symmetrical cleavage into two identical DNA strands

Question 28

How many resolvase complexes exist in eukaryotes?

Answer 28

3

Question 29

What does dissolution require?

Answer 29

1. Blm helicase 2. Top3 topoisomerase

Question 30

What is the function of topoisomerase?

Answer 30

1. removes supercoiled DNA 2. counteracts positive supercoiling during dissolution 3. creates breaks in the DNA to resolve topological issues 4. forms a phospho-tyrosine bond while cutting DNA

Question 31

How many cuts does Type II topoisomerase make?

Answer 31

2 cuts

Question 32

How many cuts does Type 1 topoisomerase make?

Answer 32

1 cute