DNA Repair

Question 1

The substrate of PARP is. . .

Answer 1

NAD⁺

Question 2

APEI endonuclease

Answer 2

Creates a 5’ single-stranded break at a site in the DNA double helix at an abasic site (presumably generated by DNA glycosylase).

Question 3

DNA of all aerobically growing cells is exposed to reactive oxygen species during normal cellular metabolism

Question 4

Mismatch repair mechanism

Answer 4

Question 5

Role of p53 in the DNA Damage Response

Answer 5

Question 6

Repair pathway overview

Answer 6

Question 7

AP lyase

Answer 7

Creates a 3’ break on an already 5’ cleaved, abasic deoxyribose, freeing the deoxyribose and leaving a completely empty space on the DNA. Acts after APEI and DNA glycosylase in base excision repair.

Question 8

Nonhomologous End Joining Mechanism

Answer 8

Question 9

KU70/80

Answer 9

The first step of nonhomologous double strand break repair.

Recognizes breaks in the chromosome and brings in proximity, then recruits repair machinery.

Question 10

DNA glycosylase

Answer 10

Flips a residue targeted for base excision repair out of the double helix and cleaves off its base, leaving the phosphodiesters and deoxyribose intact.

Question 11

Nonhomologous End Joining

Answer 11

Predominant mechanism of double strand break repair. Utilized in the early cell cycle and G0, before DNA has replicated.

DSBs are recognized by a protein complex, broken strands of DNA are aligned, and then re-ligated. Process is error prone as it does not depend on homology. Also has potential to lead to a chromosomal translocation.

Question 12

DNA Deminations

Answer 12

Deamination of cytosine and adenine represent a very common mechanism of DNA damage via hydrolysis reactions

Question 13

Direct repair mechanism

Answer 13

Question 14

Direct Repair

Answer 14

Simplest mechanism of DNA repair. An enzyme detects a base that has been damaged by addition of a functional group and removes the functional group. No further steps are taken.

Unfortunately, the utility of direct repair is limited to a small number of modifications.

Question 15

Transcription-coupled repair

Answer 15

Occurs when RNA polymerase is transcribing DNA and runs into a “bump” in the DNA that prevents it from moving. This leads to recruitment of the nucleotide excision repair complex, which repairs the lesion and allows transcription once more.

Question 16

Mismatch repair always assumes that. . .

Answer 16

. . . the daughter strand is the one with the mistake.

Question 17

CHK1 and CHK2

Answer 17

Induced by ATM and ATR. Arrest the cell cycle in two ways:

1. Phosphorylate p53, preventing Mdm2-mediated export into the cytoplasm and subsequent ubiquitin-proteasomal degradation.
2. Activate Wee1 and inhibit Cdc25, preventing the transition from G2 to M.

Question 18

Base Excision Repair and Single-Stranded Break Repair Mechanism

Answer 18

Question 19

Nucleotide excision repair

Answer 19

Removes larger lesions which are capable of distorting the overall structure of the DNA. This may include large DNA adducts spanning multiple bases or base dimerization catalyzed by UV light.

Question 20

Mismatch repair can detect _____.

Answer 20

Mismatch repair can detect all four types of possible mismatch.

Question 21

Master regulators of the DNA damage response

Answer 21

ATM and ATR

Both are protein kinases activated by MRN and KU (DNA breakage sensors), which in turn activate the distal transducers, CHK1 and CHK2, themselves kinases which induce p53-mediated cell cycle arrest.

Question 22

Mdm2

Answer 22

an E3 ubiquitin ligase for p53

Question 23

The cell’s system for recognizing kinks in DNA

Answer 23

23B and XP-C, then RPA

Question 24

Specificity in base excision repair relies on. . .

Answer 24

. . .the DNA glycosylases, which have evolved to recognize a number of different damaging DNA modifications.

Question 25

Apurinic/apyrimidinic (AP) sites

Answer 25

Most frequent type of DNA damage. Occur spontaneously by hydrolytic loss of purine or pyrimidine at a given locus.

Question 26

Poly-ADP ribose polymerase (PARP)

Answer 26

Recognizes a single stranded break in DNA (a missing residue on one side), and binds here. Generates Poly-ADP ribose at this site, a highly negatively charged polymer. This then recruits a repair complex, which removes the Poly-ADP ribose and attaches a DNA polymerase to fill in the single-nucleotide gap.

Question 27

Repair mechanisms for double-stranded breaks

Answer 27

1. **Homologous recombination** is an **error free** mechanism, but this only works if there is a sister chromatid available to help repair the lesion (only in **S or G2**) 2. **Nonhomologous end joining** operates in **G1 or G0**. This can repair the DNA, but does so with much **higher error rates**.

Question 28

Summary of Double-Stranded Break Repair Mechanisms

Answer 28

Question 29

Homologous Recombination diagram

Answer 29

Question 30

Pol β displays both ___ and \_\_\_.

Answer 30

Pol β displays both **5'→3' polymerase activity** and **3'→5' exonuclease activity**. Thus it **proofreads** its last base added as it travels along the DNA polymerizing.

Question 31

Weinberg-emphasized DNA damage → p53 induction pathway

Answer 31

Question 32

XP-C and 23B

Answer 32

Recognize lesions that distort the shape of DNA. They then recruit **TFIIH**, a helicase that unwinds the DNA at this site. **XP-G and RPA** aid in the unwinding. Finally, **XP-G and XP-F** cut ~24-32 bases upstream and downstream of the lesion, creating a gap which will be filled in by **Pol β**.

Question 33

Base Excision Repair

Answer 33

Most common mechanism of DNA repair. Recognizes a wide variety of modifications. Detects a single damaged base pair and removes one base, along with part of its phosphodiester backbone. Then, the excised base is repaired using a single base which is complimentary to the base left behind. This leaves a single-stranded break, which is then repaired by common single-strand break repair mechanisms.

Question 34

HMS-emphasized DNA damage → p53 induction pathway

Answer 34

Question 35

Mismatch repair

Answer 35

Corrects mistakes made during replication. It is **key** here that this machinery is designed to remove the mismatched base on the **daughter strand**, **NOT** **the template**.

Question 36

Homologous Recombination

Answer 36

Predominant mechanism of double strand break repair utilized in S phase and later in the cell cycle. This mechanism is nearly error-free. DSBs are recognized by a protein complex which resects DNA away from the DSB to make single-stranded regions (sticky ends). Once these are generated, resected strands invade the sister chromatid strands by identifying regions of homology. The sister chromatids are used as a template for new synthesis.

Question 37

PARP mechanism

Answer 37

Question 38

Basic DNA Repair Model

Answer 38

Question 39

Nucleotide excision repair

Answer 39

Question 40

Basic steps of DNA repair

Answer 40

1. Remove the damaged DNA 2. Fill the gap 3. Ligate the DNA

Question 41

Aflatoxin mechanism

Answer 41

Question 42

O₆-MeG vs N₇-MeG

Answer 42

Question 43

Futile cycles in nonfunctional DNA repair

Answer 43

Question 44

Lynch syndrome

Answer 44

caused by mutations in mismatch repair genes (MSH2, MSH6, MHL1), predisposes to development of colon cancer; 80% of patients carrying a mutation develop a tumor during their lifetime.

Question 45

Mutations in BRCA1 or BRCA2 genes

Answer 45

involved in homologous recombination, predispose to breast and ovarian cancer; 30-80% of patients with a mutation will develop breast or ovarian cancer, depending on which gene is mutated

Question 46

Mutations in XP

Answer 46

Xeroderma pigmentosum is due to mutation in nucleotide excision repair.

Question 47

Direct repair enzymes tend to. . .

Answer 47

. . .**steal and sequester** modifications. They are not a type of enzyme which may replenish itself and continue to catalyze the reaction, it is a one-to-one ratio. Once the resource has been exhausted, mutations in DNA will accumulate.

Question 48

Types of kinetochore attachment

Answer 48