Ali Questions Flashcards
Why ataxia-telangiectasia patiens are more sensitive to radiation?
Defective ATM
which is an improtant DNA damage kinase.
DNA Repair I - Floyd
What are the sources of DNA damage?
- endogenous agents (~500K events/day):
- Metabolism byproducts (base damage)
- Free radicals (base damage, strand breaks) - exogenous Agents
- ionizing radiation (all kinds: bulky, base, strand breaks)
- chemicals (all kinds: alkylation, strand breaks) - Normal processes
- immune system diversity (double strand breaks, mismatch, and base excision)
- Meiosis (double strand breaks)
- replication problems (mismatch error, stalled forks, strand breaks)
DNA Repair I - Floyd
Radiation kills tumor cells by which types of DNA damages?
Which one is the most lethal?
- Breaks:
SSBs
DSBs - Base damages:
change
loss (abasic sites) - crosslinks
DNA-DNA
DNA-protein
DNA Repair I - Floyd
Approximately how many DSBs/cell/Gy of radiation happens?
30-50 DSBs
compare to:
~1K SSBs, ~1K sugar damage, ~1K-2K base damage, loss or substitution
~100-200 DNA-protein crosslinks
~30 DNA-DNA crosslinks
DNA Repair I - Floyd
What are DNA damage repair pathways in response to double strand breaks?
Which one is error prone?
When in the cell cycle which one happens?
- Non-homologous end joining (NHEJ)
- error-prone
- G0/G1 phases of cell cylce (no sister chromatid) - Homologous recombination repair (HRR)
- “high fidelity” template directed
- S/G2 phases of cell cycle - Microhomology-Medicated End Joining (MMEJ)
DNA Repair I - Floyd
Which proteins are involved in NHEJ?
Ku70/Ku80
DNApk
Artemis
ATM
gamma H2AX
DNA Repair I - Floyd
Which proteins are involved in homologous recombination?
ATM
BRCA1, BRCA2
RAD51
DNA repair I - Floyd
What are Holiday junctions and they are formed durign what type of DNA repair?
Homologous recombination
DNA repair I - Floyd
Which enzyme performs 5’ -> 3’ resection during homologous recombination?
MRX
DNA repair I - Floyd
What is patients with xeroderma pigmentosa are sensitive to? Why?
Ultraviolet light
DNA damage repair dysfunction that distorts the helix.
Nucleotide excision repair (NER)
DNA repair I - Floyd
What are the different types of single strand break repairs? Which lesions each one repair?
- Base excision repair (BER)
oxidized, alkylated, deaminated bases, or uracil - Nucleotide excisino repair (NER)
cyclobutane pyrimidine dimers, photoproducts - Mismatch repair
repair of G/T and A/C pairs
DNA repair I - Floyd
What are the key molecules involved in these parts of NHEJ?
- Mistake recognition
- Mistake excision
- Mistake repair
- Mistake recognition
Ku70/Ku80
DNA-PKcs - Mistake excision
DNA-PKcs
Artemis - Mistake repair
Pol gamm/mu
XRCC4
Ligase IV
DNA repair I - Floyd
What are the key molecules involved in these parts of homologus recombination?
- Mistake recognition
- Mistake excision
- Mistake repair
- Mistake recognition
MRN complex
FANC proteines - Mistake excision
ATM
BRCA1/BARD1
MRN complex
RPA
PALB2
BRCA2
RAD51
CtlP - Mistake repair
DNA polymerases
DNA repair I - Floyd
What are the key molecules involved in these parts of mismatch repair?
- Mistake recognition
- Mistake excision
- Mistake repair
- Mistake recognition
MutSx (MSH2, MSH6) - Mistake excision
MutLx (MLH1/PMS2)
exonuclease (EXO1) - Mistake repair
Pol gamma
PCNA
RPA
DNA ligase
DNA repair I - Floyd
What are the key molecules involved in these parts of nucleotide excision repair?
- Mistake recognition
- Mistake excision
- Mistake repair
- Mistake recognition
RNA polymerase I and II
CSA and CSB - Mistake excision
TFIIH
XPD, XPG, XPF-ERCC1
5’nuclease
RPA - Mistake repair
PCNA
Poly gamma/epsilon/kappa
DNA ligase I or III
XRCC1
DNA repair I - Floyd
What are the key molecules involved in these parts of base excision repair?
- Mistake recognition
- Mistake excision
- Mistake repair
- Mistake recognition
DNA glycosylase (MYH)
AP lyase - Mistake excision
APE 1
AP lyase
PARP1
XRCC1
Fen1
PCNA
PolyB
Pol gamma/epsilon - Mistake repair
XRCC1
PolB
Fen1
DNA ligase I
DNA ligase III
DNA repair I - Floyd
Which genetic diseases are associated with defects in base excision repair?
Often embryologically lethal
POLB mutations found in 30% of human cancers
MYH mutations increase susceptibility to colon cancer
DNA repair I - Floyd
Which genetic diseases are associated with defects in nucleotide excision repair?
Xeroderma pigmentosum (XP gene mutations)
DNA repair I - Floyd
Which genetic diseases are associated with defects in mismatch repair?
Lynch syndrome (hereditary nonpolyposis colorectal cancer) (MSH2 and MLH1)
DNA repair I - Floyd
Which genetic diseases are associated with defects in homologous recombination?
BRCA1/2 mutations
Fanconi anemia (FANC genes)
ataxia-telangiectasia (ATM)
DNA repair I - Floyd
Which genetic diseases are associated with defects in non-homologous end joining?
LIG4 syndrome (LIG4)
XLF-SCID (XLF)
Name two assays to measure cell proliferation.
Tritiated Thymidine (3H-TdR) and Bromodeoxyuridine (BrdU) Assays
These nucleoside analogues incorporate into DNA in proliferating cell populations
Higher levels = higher proliferation
Cell cycle
Which lab method is used for synchronization of the cell population?
mitotic shakeoff - enriches for mitotic cells
Thymidine block (or double thymidine block) - blocks cells at G1/S
Cell cycle
Which part of the cell cycle is the most variable (most regulated)?
G1
Cell cycle
Which part of the cell cycle, stem cells are in mostly?
G0
Cell cycle
What does cell cycle analysis do?
Stain DNA with propidium iodide
flow -> quantify fluorescent signal
allows discremination between N (pre-S) and 2N (post-S)
know the graph
Cell cycle
What is the difference between G0 of normal and cancer cells?
Cancer cells usually don’t enter G0 unless something happens
Normal cells - physiologically enter GO as a normal process
* Tumor cells - most often don’t enter GO unless some stimulus induces it (low growth factors, cell-cell contact inhibition)
Cell cycle
Phospho-histone H3 and Ki67 stains are higher in which parts of the cell cycle?
Phospho-Histone H3 - high in S phase
Ki67 - high in G2/M phase
Roughly correlate to the number of cells proliferating
Cell cycle
Which cyclins are most active in which parts of the cell cycle?
DEAB
D: G1
E:S
A:G2
B:M
Cell cycle
Which cyclin/CDKs are associated with which parts of the cell cycle?
D/CDK 4/6: G1 till R point
E/CDK 2: R point - early S
A/CDK2 - early S
A/CDC2 - S/G2
B/CDC2 - M
Cell cycle
What does pimonidazole do?
Detects hypoxia
Hypoxia
