DNA Damage and Repair

Question 1

What is the size of a ‘spur’ and how many ionization events?

Answer 1

4 nm in diameter

3 ion pairs

Question 2

What is the size of a ‘blob’ and how many ionization events?

Answer 2

7 nm

12 ion pairs

Question 3

What is the approximate width of dsDNA? length of a turn?

Answer 3

2 nm

3.4 nm

Question 4

What predominates with exposure to low LET? high LET? (blobs vs. spurs)

Answer 4

low LET is sparsely ionizing, spurs predominate

high LET is densely ionizing, blobs predominate

Question 5

How common is the following damage/cell with 1Gy?

1) ionization events
2) base damage
3) SSBs
4) DSBs
5) cell death

Answer 5

1) 100k
2) >5k
3) 1k
4) 40
5) 0.63 (SF = 0.37)

Question 6

How does a dicentric chromosome form?

Answer 6

Two separate chromatids have a DSB, the free ends fuse –> p1/centromere/q1p2/centromere/q2

Question 7

How does a ring form?

Answer 7

The same chromatid has DSB on both ends, the free ends circularize

Question 8

How does an anaphase bridge form?

Answer 8

Sister chromatids on the same chromosome have DSB, the two sister free ends fuse. These are unable to segregate nicely during anaphase, and random breaks are induced (with loss of DNA in a daughter)

Question 9

Approximately what total body dose is needed to see chromosome aberrations in lymphocytes?

Answer 9

> 0.2 Gy. Above 4 Gy, lymphocytes undergo apoptosis and chromosome damage is not seen

Question 10

What is the process of base excision repair?

Answer 10

Involved proteins: APE1, XRCC1, PARP, PolB, Lig3

1) APE1 and PARP interact with the site to remove the base/backbone
2) XRCC1 stabilizes the induced break while PolB repairs it
3) Ligase3 seals the backbone repair

Question 11

What is the process of nucleotide excision repair?

Answer 11

Involved proteins: XP family, RPA, ERCC1, CSA, polD, Lig1

1) XPA proteins and CSA recognize the lesion
2) XPA and RPA stabilize the ssDNA ‘bubble’
3) ERCC1 and XPG excise the segment
4) Pol-delta synthesizes the repair strand
5) Ligase I seals the backbone

Question 12

When is NER utilized vs BER?

Answer 12

BER is for small, single base disruptions whereas NER is for larger, bulky lesions (UV, alkyl groups, platinum)

Question 13

What is the process of MMR?

Answer 13

Involved proteins: MSH, MLH, Exo1, polD, Lig1

1) MSH and MLH recognize/stabilize the mismatch
2) Exo1 removes the lesion
3) RPA stabilizes the ssDNA while pol-delta synthesizes
4) Lig1 seals backbone

Essentially the same ‘repair’ steps as NER

Question 14

What causes a defect in MMR? What does this cause?

Answer 14

Lynch syndrome

Microsatellite instability

Question 15

What is the initiation of DSB recognition and repair?

Answer 15

ATM and ATR recognize the DSBs, phosphorylate gH2ax, and recruit the MRN complex (Mre11, Rad50, Nbs1)

Then, there is a competition to undergo HRR or NHEJ, facilitated by 53BP1

p53 and Chk1/Chk2 are activated as well

Question 16

What is the function of ATM?

Answer 16

Recognition of DSB

Regulation of MRN activity (resection of ends)

Question 17

What is the function of 53BP1?

Answer 17

53BP1 inhibits the exonuclease activity of the MRN complex, prohibiting HRR

Question 18

What is the NHEJ pathway?

Answer 18

Key proteins: Ku70, Ku80, Artemis, PKcs, Pol-lambda

1) Ku70/Ku80 dimer binds free ends
2) DNA-PKcs is recruited to juxtapose the free ends
3) Artemis removes overhangs
4) XRCC4/Pol-L/Lig4 then ligate the free ends

Question 19

When does NHEJ predominate?

Answer 19

during G0/G1, when there is no sister chromatid as a template

Question 20

What is the HRR pathway?

Answer 20

Key proteins: MRN complex, RPA, Rad51, Rad52, BRCA1/2,

1) ATM recruits the MRN complex (rad50) and BRCA1 to the site of damage
2) Mre11 creates a 3’ ssDNA overhang
3) BRCA2 facilitates the recruitment of RPA to stabilize the ssDNA and rad51 to allow strand invasion of the sister chromatid
4) Rad52 then removes rad51 after invasion to allow DNA duplex formation
5) A helicase helps unwind the template and pol synthesizes through the Holliday junction

Question 21

What disease is associated with NER deficiency?

Answer 21

Xeroderma Pigmentosum

Question 22

What disease is associated with CSA/CSB deficiency? What path is this in?

Answer 22

Cockayne Syndrome (NER)
Photosensitivity without cancer risk

Question 23

What is disrupted in Lynch Syndrome?

Answer 23

MSH/MLH

Question 24

Are BRCA1/2 patients radiosensitive?

Answer 24

Surprisingly no

Question 25

What happens in ATM disorders?

Answer 25

extremely radiosensitive
high risk of multiple cancers
neuro/immuno deficits

Question 26

What is disrupted in ATM-like disorder?

Answer 26

Mre11

Question 27

What is disrupted in Niemann Breakage Syndrome?

Answer 27

Nbs1

Question 28

What is the pathway of crosslink repair in S phase?

Answer 28

1) crosslink is recognized by FANCM at stalled replication forks, which recruits the FA core complex
2) FAN1 and XPF excise the crosslinked base backbone (thus creating a ssDNA break in a replication bubble, effectively a dsDNA break)
3) Rev1/Pol-E continue synthesis across the lesion
4) the dsDNA break is repaired by HRR
5) NER excises the remainder of the crosslink

Question 29

What is associated with Fanconi anemia?

Answer 29

loss of crosslink repair; leukemias

Question 30

What is lost in Bloom Syndrome? What is seen?

Answer 30

A RecQ helicase (BLM)

GI tumors, sunlight sensitivity, dwarfism, elevated sister chromatid exchanges

Question 31

What is lost in Warner Syndrome? What is seen?

Answer 31

A RecQ helicase (WS)

Premature aging, early cancers

Question 32

What is Usher Syndrome?

Answer 32

loss of hearing and sight

very radiosensitive

Question 33

What is Gardner Syndrome?

Answer 33

mutation in APC –> colon cancers

Question 34

What is basal cell nevoid syndrome?

Answer 34

aka Gorlin syndrome
mutation in PTCH tumor suppressor
basal cells, odontogenic tumor