Hall 2 - Molecular Mechanisms of DNA & Chromosome Damage & Repair Flashcards
How many DSBs after 1 Gy?
40
How many SSBs after 1 Gy?
1000
How many bases damaged after 1 Gy?
> 2000
How many DNA-DNA crosslinks after 1 Gy?
30
What type of damage predominates in cell killing?
Double strand breaks
Creates chromosomal aberrations that are problematic at mitosis
What is the energy of a spur?
<100 eV
~3 ion pairs
4nm diameter
Most common for photons
What is the energy of a blob?
100-500 eV
~12 ion pairs
7nm diameter
Densely ionizing radiation
What is the energy of a short track?
500-5000 eV
What happens when spurs and blobs cluster/ multiple sites are damaged in the same area?
Locally multiply damaged site
What is used to lyse cells and run the DNA on an agarose gel to separate according to size?
Pulsed-field gel electrophoresis (PFGE)
Fraction of DNA released is directly proportional to dose
What is used to lyse cells and see how much damaged DNA migrates (as fragments)?
Single-cell electrophoresis (comet assay)
Offers single-cell resolution
What type of lysis buffer assays for DSBs?
Neutral
What type of lysis buffer assays for SSBs?
Alkaline
What is an antibody that can be used to detect foci that contain signaling/repair proteins (DNA damage-induced nuclear foci) for DSBs?
gamma-H2AX
(Ab to phosphorylated H2AX)
or
53BP1
(only found at damaged loci)
What phosphorylates H2AX? When?
ATM
After DSBs
What can be used to measure radiosensitivity 24 hours after RT?
Amount of gamma-H2AX
Reflects extent of repair (detects H2AX phosphorylated by ATM after DSB, so high levels mean poor repair)
What repair mechanism is used when there is an incorrect base in the DNA?
Base excision repair
What initially binds to the site of a single strand break?
PARP and XRCC1
Remaining process is like BER
What removes bulky adducts in DNA?
Nucleotide excision repair
What is the damage recognition complex in NER?
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
What senses mismatch repair?
MSH2/6 or MSH2/3 complex
Recruits MLH1, PMS1/2, MLH3
What type of DNA double-strand break repair is error free? When does it occur in the cell cycle?
Homologous recombination
Requires an undamaged DNA strand (sister chromatid) to serve as template
Error-free
Occurs in late S and G2 phase ONLY
What type of DNA double-strand break repair occurs in G1?
Non-homologous end-joining
Error-prone because no sequence homology
What is the primary responder and orchestrator of DSBs?
ATM
For both HR and NHEJ:
ATM/MRN complex (Mre11/Rad50/Nbs1) binds initially
What regulates HR vs NHEJ?
P53BP1
Inhibits HR
After ATM/MRN complex binds in NHEJ, what proteins are recruited?
Ku70/Ku80 heterodimer
NHEJ:
ATM/MRN»_space; Ku70/Ku80
» recruits DNA-PKcs
» end processing by Artemis
» repair synthesis and ligation by XRCC4
What does the end processing in NHEJ?
Artemis
(endonuclease)
NHEJ:
ATM/MRN»_space; Ku70/Ku80
» recruits DNA-PKcs
» end processing by Artemis
» repair synthesis and ligation by XRCC4
What ligates at the end of NHEJ?
XRCC4
NHEJ:
ATM/MRN»_space; Ku70/Ku80
» recruits DNA-PKcs
» end processing by Artemis
» repair synthesis and ligation by XRCC4
After ATM/MRN complex binds in HR, what proteins are recruited?
RPA and BRCA1/2
HR:
ATM/MRN»_space; RPA and BRCA1/2
» loads RAD51 onto RPA (RAD52 protects from degradation)
» strand invasion
» Holliday junction
» resolved via crossover or non-crossover
What catalyzes strand invasion in HR?
RAD51
HR:
ATM/MRN»_space; RPA and BRCA1/2
» loads RAD51 onto RPA (RAD52 protects from degradation)
» strand invasion
» Holliday junction
» resolved via crossover or non-crossover
What happens when HR is dysregulated?
Loss of heterozygosity (LOH)
» cancer
What recognizes interstrand crosslink repair complexes in S phase?
Fanconi anemia complex
Recognizes replication fork stalling
Excises and repairs with HR and NER
What pathology leads to hypersensitivity to crosslinking agents?
Fanconi anemia
Resulting abberations = quadriradials
Cells with defects in NER or HR are moderately sensitive
What type of damage is caused by UV light and how is it repaired?
Pyrimidine dimers, 6-4 photoproducts
NER
What type of damage is caused by ionizing RT and how is it repaired?
DSBs, SSBs, base damage, DNA-histone crosslinks
DSB repair (HR, NHEJ), BER
What do BER defects lead to?
Increased mutation rate (not radiosensitivity)
What do NER defects lead to?
Increased sensitivity to UV light (XP) and chemo that creates bulky adducts (alkylating agents)
Does not increase radiosensitivity
What do DSB repair defects lead to?
Increased radiosensitivity
NHEJ > HR
NHEJ defects also causes immunodeficiency due to impaired VDJ recombination
What do defects in MSH, MLH, or PMS proteins lead to?
HNPCC/Lynch syndrome
What happens in prophase?
Chromatin condensation, centromere visible
What happens in metaphase?
Spindle formation and chromosome alignment in center
What happens in anaphase?
Chromosomes move to poles
What happens in telophase?
Daughter cells pull apart, chromosomes uncoil, nuclear membrane reappears
When do chromosome aberrations happen?
Before duplication (G1)
Duplicated»_space; identical aberrations in both sister chromatids
When do chromatid aberrations happen?
After duplication (late S, G2)
Only one sister chromatid injured
What are three lethal aberrations?
Dicentrics (pre-replication)
Ring (pre-replication)
Anaphase bridge (post-replication)
Require two chromosome DSBs
What are two non-lethal aberrations?
Symmetric translocation (pre-replication)
Small interstitial deletion
Why are micronuclei prognostic of cell death?
Pseudo-nuclear membranes that envelope DNA lacking a centromere (acentric fragments)»_space; loss of genetic material at next mitosis
What pathway do micronuclei activate?
cGAS-STING
> > INF-beta
infiltration of tumor by CD8+ T cells
What causes the linear part of the cell killing curve?
Single charged particle track creates two chromosome DSBs (low doses)
*Terminal deletions result from one DSB, therefore also linear function of dose
What causes the quadratic part of the cell killing curve?
2 separate radiation tracks (high doses)
When can chromosomal aberrations be seen?
During mitosis (condensed chromatin)
Exception: micronuclei (indicate acentrics, which can be detected in interphase)
What is the lowest single dose of RT that can be detected?
0.25 Gy
What is a stable vs unstable aberration?
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
