Ali Questions

Question 1

Why ataxia-telangiectasia patiens are more sensitive to radiation?

Answer 1

Defective ATM
which is an improtant DNA damage kinase.

DNA Repair I - Floyd

Question 2

What are the sources of DNA damage?

Answer 2

1. endogenous agents (~500K events/day):
   - Metabolism byproducts (base damage)
   - Free radicals (base damage, strand breaks)
2. exogenous Agents
   - ionizing radiation (all kinds: bulky, base, strand breaks)
   - chemicals (all kinds: alkylation, strand breaks)
3. 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

Question 3

Radiation kills tumor cells by which types of DNA damages?
Which one is the most lethal?

Answer 3

1. Breaks:
   SSBs
   DSBs
2. Base damages:
   change
   loss (abasic sites)
3. crosslinks
   DNA-DNA
   DNA-protein

DNA Repair I - Floyd

Question 4

Approximately how many DSBs/cell/Gy of radiation happens?

Answer 4

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

Question 5

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?

Answer 5

1. Non-homologous end joining (NHEJ)
   - error-prone
   - G0/G1 phases of cell cylce (no sister chromatid)
2. Homologous recombination repair (HRR)
   - “high fidelity” template directed
   - S/G2 phases of cell cycle
3. Microhomology-Medicated End Joining (MMEJ)

DNA Repair I - Floyd

Question 6

Which proteins are involved in NHEJ?

Answer 6

Ku70/Ku80
DNApk
Artemis
ATM
gamma H2AX

DNA Repair I - Floyd

Question 7

Which proteins are involved in homologous recombination?

Answer 7

ATM
BRCA1, BRCA2
RAD51

DNA repair I - Floyd

Question 8

What are Holiday junctions and they are formed durign what type of DNA repair?

Answer 8

Homologous recombination

DNA repair I - Floyd

Question 9

Which enzyme performs 5’ -> 3’ resection during homologous recombination?

Answer 9

MRX

DNA repair I - Floyd

Question 10

What is patients with xeroderma pigmentosa are sensitive to? Why?

Answer 10

Ultraviolet light
DNA damage repair dysfunction that distorts the helix.
Nucleotide excision repair (NER)

DNA repair I - Floyd

Question 11

What are the different types of single strand break repairs? Which lesions each one repair?

Answer 11

1. Base excision repair (BER)
   oxidized, alkylated, deaminated bases, or uracil
2. Nucleotide excisino repair (NER)
   cyclobutane pyrimidine dimers, photoproducts
3. Mismatch repair
   repair of G/T and A/C pairs

DNA repair I - Floyd

Question 12

What are the key molecules involved in these parts of NHEJ?

1. Mistake recognition
2. Mistake excision
3. Mistake repair

Answer 12

1. Mistake recognition
   Ku70/Ku80
   DNA-PKcs
2. Mistake excision
   DNA-PKcs
   Artemis
3. Mistake repair
   Pol gamm/mu
   XRCC4
   Ligase IV

DNA repair I - Floyd

Question 13

What are the key molecules involved in these parts of homologus recombination?

1. Mistake recognition
2. Mistake excision
3. Mistake repair

Answer 13

1. Mistake recognition
   MRN complex
   FANC proteines
2. Mistake excision
   ATM
   BRCA1/BARD1
   MRN complex
   RPA
   PALB2
   BRCA2
   RAD51
   CtlP
3. Mistake repair
   DNA polymerases

DNA repair I - Floyd

Question 14

What are the key molecules involved in these parts of mismatch repair?

1. Mistake recognition
2. Mistake excision
3. Mistake repair

Answer 14

1. Mistake recognition
   MutSx (MSH2, MSH6)
2. Mistake excision
   MutLx (MLH1/PMS2)
   exonuclease (EXO1)
3. Mistake repair
   Pol gamma
   PCNA
   RPA
   DNA ligase

DNA repair I - Floyd

Question 15

What are the key molecules involved in these parts of nucleotide excision repair?

1. Mistake recognition
2. Mistake excision
3. Mistake repair

Answer 15

1. Mistake recognition
   RNA polymerase I and II
   CSA and CSB
2. Mistake excision
   TFIIH
   XPD, XPG, XPF-ERCC1
   5’nuclease
   RPA
3. Mistake repair
   PCNA
   Poly gamma/epsilon/kappa
   DNA ligase I or III
   XRCC1

DNA repair I - Floyd

Question 16

What are the key molecules involved in these parts of base excision repair?

1. Mistake recognition
2. Mistake excision
3. Mistake repair

Answer 16

1. Mistake recognition
   DNA glycosylase (MYH)
   AP lyase
2. Mistake excision
   APE 1
   AP lyase
   PARP1
   XRCC1
   Fen1
   PCNA
   PolyB
   Pol gamma/epsilon
3. Mistake repair
   XRCC1
   PolB
   Fen1
   DNA ligase I
   DNA ligase III

DNA repair I - Floyd

Question 17

Which genetic diseases are associated with defects in base excision repair?

Answer 17

Often embryologically lethal
POLB mutations found in 30% of human cancers
MYH mutations increase susceptibility to colon cancer

DNA repair I - Floyd

Question 18

Which genetic diseases are associated with defects in nucleotide excision repair?

Answer 18

Xeroderma pigmentosum (XP gene mutations)

DNA repair I - Floyd

Question 19

Which genetic diseases are associated with defects in mismatch repair?

Answer 19

Lynch syndrome (hereditary nonpolyposis colorectal cancer) (MSH2 and MLH1)

DNA repair I - Floyd

Question 20

Which genetic diseases are associated with defects in homologous recombination?

Answer 20

BRCA1/2 mutations
Fanconi anemia (FANC genes)
ataxia-telangiectasia (ATM)

DNA repair I - Floyd

Question 21

Which genetic diseases are associated with defects in non-homologous end joining?

Answer 21

LIG4 syndrome (LIG4)
XLF-SCID (XLF)

Question 22

Name two assays to measure cell proliferation.

Answer 22

Tritiated Thymidine (3H-TdR) and Bromodeoxyuridine (BrdU) Assays

These nucleoside analogues incorporate into DNA in proliferating cell populations
Higher levels = higher proliferation

Cell cycle

Question 23

Which lab method is used for synchronization of the cell population?

Answer 23

mitotic shakeoff - enriches for mitotic cells
Thymidine block (or double thymidine block) - blocks cells at G1/S

Cell cycle

Question 24

Which part of the cell cycle is the most variable (most regulated)?

Answer 24

G1

Cell cycle

Question 25

Which part of the cell cycle, stem cells are in mostly?

Answer 25

G0 ## Footnote Cell cycle

Question 26

What does cell cycle analysis do?

Answer 26

Stain DNA with propidium iodide flow -> quantify fluorescent signal allows discremination between N (pre-S) and 2N (post-S) know the graph ## Footnote Cell cycle

Question 27

What is the difference between G0 of normal and cancer cells?

Answer 27

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) ## Footnote Cell cycle

Question 28

Phospho-histone H3 and Ki67 stains are higher in which parts of the cell cycle?

Answer 28

Phospho-Histone H3 - high in S phase Ki67 - high in G2/M phase Roughly correlate to the number of cells proliferating ## Footnote Cell cycle

Question 29

Which cyclins are most active in which parts of the cell cycle?

Answer 29

DEAB D: G1 E:S A:G2 B:M ## Footnote Cell cycle

Question 30

Which cyclin/CDKs are associated with which parts of the cell cycle?

Answer 30

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 ## Footnote Cell cycle

Question 31

What does pimonidazole do?

Answer 31

Detects hypoxia ## Footnote Hypoxia