Hall 2 - Molecular Mechanisms of DNA & Chromosome Damage & Repair

Question 1

How many DSBs after 1 Gy?

Answer 1

40

Question 2

How many SSBs after 1 Gy?

Answer 2

1000

Question 3

How many bases damaged after 1 Gy?

Answer 3

> 2000

Question 4

How many DNA-DNA crosslinks after 1 Gy?

Answer 4

30

Question 5

What type of damage predominates in cell killing?

Answer 5

Double strand breaks

Creates chromosomal aberrations that are problematic at mitosis

Question 6

What is the energy of a spur?

Answer 6

<100 eV

~3 ion pairs
4nm diameter
Most common for photons

Question 7

What is the energy of a blob?

Answer 7

100-500 eV

~12 ion pairs
7nm diameter
Densely ionizing radiation

Question 8

What is the energy of a short track?

Answer 8

500-5000 eV

Question 9

What happens when spurs and blobs cluster/ multiple sites are damaged in the same area?

Answer 9

Locally multiply damaged site

Question 10

What is used to lyse cells and run the DNA on an agarose gel to separate according to size?

Answer 10

Pulsed-field gel electrophoresis (PFGE)

Fraction of DNA released is directly proportional to dose

Question 11

What is used to lyse cells and see how much damaged DNA migrates (as fragments)?

Answer 11

Single-cell electrophoresis (comet assay)

Offers single-cell resolution

Question 12

What type of lysis buffer assays for DSBs?

Answer 12

Neutral

Question 13

What type of lysis buffer assays for SSBs?

Answer 13

Alkaline

Question 14

What is an antibody that can be used to detect foci that contain signaling/repair proteins (DNA damage-induced nuclear foci) for DSBs?

Answer 14

gamma-H2AX
(Ab to phosphorylated H2AX)

or

53BP1
(only found at damaged loci)

Question 15

What phosphorylates H2AX? When?

Answer 15

ATM

After DSBs

Question 16

What can be used to measure radiosensitivity 24 hours after RT?

Answer 16

Amount of gamma-H2AX

Reflects extent of repair (detects H2AX phosphorylated by ATM after DSB, so high levels mean poor repair)

Question 17

What repair mechanism is used when there is an incorrect base in the DNA?

Answer 17

Base excision repair

Question 18

What initially binds to the site of a single strand break?

Answer 18

PARP and XRCC1

Remaining process is like BER

Question 19

What removes bulky adducts in DNA?

Answer 19

Nucleotide excision repair

Question 20

What is the damage recognition complex in NER?

Answer 20

XPC/XPE in non-transcribed region (global)
CSA/CSB in transcribed region

*Any protein starting with “XP” is named for its involvement in xeroderma pigmentosum

**CSA/B = Cockayne syndrome

Question 21

What senses mismatch repair?

Answer 21

MSH2/6 or MSH2/3 complex

Recruits MLH1, PMS1/2, MLH3

Question 22

What type of DNA double-strand break repair is error free? When does it occur in the cell cycle?

Answer 22

Homologous recombination

Requires an undamaged DNA strand (sister chromatid) to serve as template

Error-free

Occurs in late S and G2 phase ONLY

Question 23

What type of DNA double-strand break repair occurs in G1?

Answer 23

Non-homologous end-joining

Error-prone because no sequence homology

Question 24

What is the primary responder and orchestrator of DSBs?

Answer 24

ATM

For both HR and NHEJ:
ATM/MRN complex (Mre11/Rad50/Nbs1) binds initially

Question 25

What regulates HR vs NHEJ?

Answer 25

P53BP1 Inhibits HR

Question 26

After ATM/MRN complex binds in NHEJ, what proteins are recruited?

Answer 26

Ku70/Ku80 heterodimer NHEJ: ATM/MRN >> Ku70/Ku80 >> recruits DNA-PKcs >> end processing by Artemis >> repair synthesis and ligation by XRCC4

Question 27

What does the end processing in NHEJ?

Answer 27

Artemis (endonuclease) NHEJ: ATM/MRN >> Ku70/Ku80 >> recruits DNA-PKcs >> end processing by Artemis >> repair synthesis and ligation by XRCC4

Question 28

What ligates at the end of NHEJ?

Answer 28

XRCC4 NHEJ: ATM/MRN >> Ku70/Ku80 >> recruits DNA-PKcs >> end processing by Artemis >> repair synthesis and ligation by XRCC4

Question 29

After ATM/MRN complex binds in HR, what proteins are recruited?

Answer 29

RPA and BRCA1/2 HR: ATM/MRN >> RPA and BRCA1/2 >> loads RAD51 onto RPA (RAD52 protects from degradation) >> strand invasion >> Holliday junction >> resolved via crossover or non-crossover

Question 30

What catalyzes strand invasion in HR?

Answer 30

RAD51 HR: ATM/MRN >> RPA and BRCA1/2 >> loads RAD51 onto RPA (RAD52 protects from degradation) >> strand invasion >> Holliday junction >> resolved via crossover or non-crossover

Question 31

What happens when HR is dysregulated?

Answer 31

Loss of heterozygosity (LOH) >> cancer

Question 32

What recognizes interstrand crosslink repair complexes in S phase?

Answer 32

Fanconi anemia complex Recognizes replication fork stalling Excises and repairs with HR and NER

Question 33

What pathology leads to hypersensitivity to crosslinking agents?

Answer 33

Fanconi anemia Resulting abberations = quadriradials Cells with defects in NER or HR are moderately sensitive

Question 34

What type of damage is caused by UV light and how is it repaired?

Answer 34

Pyrimidine dimers, 6-4 photoproducts NER

Question 35

What type of damage is caused by ionizing RT and how is it repaired?

Answer 35

DSBs, SSBs, base damage, DNA-histone crosslinks DSB repair (HR, NHEJ), BER

Question 36

What do BER defects lead to?

Answer 36

Increased mutation rate (not radiosensitivity)

Question 37

What do NER defects lead to?

Answer 37

Increased sensitivity to UV light (XP) and chemo that creates bulky adducts (alkylating agents) Does not increase radiosensitivity

Question 38

What do DSB repair defects lead to?

Answer 38

Increased radiosensitivity NHEJ > HR NHEJ defects also causes immunodeficiency due to impaired VDJ recombination

Question 39

What do defects in MSH, MLH, or PMS proteins lead to?

Answer 39

HNPCC/Lynch syndrome

Question 40

What happens in prophase?

Answer 40

Chromatin condensation, centromere visible

Question 41

What happens in metaphase?

Answer 41

Spindle formation and chromosome alignment in center

Question 42

What happens in anaphase?

Answer 42

Chromosomes move to poles

Question 43

What happens in telophase?

Answer 43

Daughter cells pull apart, chromosomes uncoil, nuclear membrane reappears

Question 44

When do chromosome aberrations happen?

Answer 44

Before duplication (G1) Duplicated >> identical aberrations in both sister chromatids

Question 45

When do chromatid aberrations happen?

Answer 45

After duplication (late S, G2) Only one sister chromatid injured

Question 46

What are three lethal aberrations?

Answer 46

Dicentrics (pre-replication) Ring (pre-replication) Anaphase bridge (post-replication) Require two chromosome DSBs

Question 47

What are two non-lethal aberrations?

Answer 47

Symmetric translocation (pre-replication) Small interstitial deletion

Question 48

Why are micronuclei prognostic of cell death?

Answer 48

Pseudo-nuclear membranes that envelope DNA lacking a centromere (acentric fragments) >> loss of genetic material at next mitosis

Question 49

What pathway do micronuclei activate?

Answer 49

cGAS-STING >> INF-beta >> infiltration of tumor by CD8+ T cells

Question 50

What causes the linear part of the cell killing curve?

Answer 50

Single charged particle track creates two chromosome DSBs (low doses) *Terminal deletions result from one DSB, therefore also linear function of dose

Question 51

What causes the quadratic part of the cell killing curve?

Answer 51

2 separate radiation tracks (high doses)

Question 52

When can chromosomal aberrations be seen?

Answer 52

During mitosis (condensed chromatin) Exception: micronuclei (indicate acentrics, which can be detected in interphase)

Question 53

What is the lowest single dose of RT that can be detected?

Answer 53

0.25 Gy

Question 54

What is a stable vs unstable aberration?

Answer 54

Stable = translocations (last for years, persist in stem cells) Unstable = rings/dicentrics (disappear over time, only in terminally differentiated lymphocytes) *Lymphocytes disappear quickly at high doses