DNA Repair

Question 1

What causes DNA mutations?

Answer 1

Mutations are the result of replication errors.

Question 2

What are the mechanisms that cause DNA lesions?

Answer 2

Lesions arise via Intrinsic and Extrinsic mechanisms.

Question 3

Intrinsic Mechanisms

(of DNA damage)

Answer 3

Misincorporation of nucleotide by DNA polymerase during replication.

Question 4

Extrinsic Mechanisms

(of DNA damage)

Answer 4

DNA damage by radiation, chemicals, or other agents leading to misincorporation during replication.

*ROS may lead to ~200,000 lesions/cell/day

Question 5

Describe the significance of DNA damage.

(3 important points)

Answer 5

1. DNA damage is associated with cancer and aging.
2. A fraction of DNA damage escapes repair and mutations can accumulate, leading to cell death, senescence, or cellular dysfunction.
3. Some DNA damage is mutagenic and can lead to cancer. Other types of damage (dsDNA breaks, DNA crosslinking) are not as mutagenic but lead to tissue degeneration and aging.

Question 6

How can a DNA lesion lead to a mutation?

Answer 6

Some types of lesions participate in altered base-pairing during replication. This nucleotide misincorporation during replication can lead to a mutation.

Question 7

What are the two broad types of DNA repair pathways? Give examples for each.

Answer 7

1. Pathways that repair damaged bases (ie direct base repair, BER, and NER).
2. Pathways that repair damaged phosphodiester bonds / double-stranded breaks (ie homologous recombination repair and non-homologous end joining).

Question 8

Describe the excision repair pathways.

Answer 8

The damaged or incorrect base is removed.

Question 9

Compare the BER and NER pathways.

Answer 9

Base excision repair (BER) - the defective base is removed, resulting in an “AP” site. Corrects “non-bulky” DNA damage.

Nucleotide excision repair (NER) - a stretch of the damaged strand is removed and replaced by DNA synthesis.

Question 10

Describe Base Excision Repair (BER)

Answer 10

BER corrects “non-bulky” DNA damage using a DNA glycosylase. DNA glycosylases recognize a damaged base, remove them by hydrolyzing the glycosidic bond, and generate an AP site.

Question 11

What is the main pathway of Base Excision Repair?

Answer 11

Short patch repair, replacement of a damaged base with a single nucleotide, represents ~80-90% of BER.

Question 12

What is the backup pathway of BER?

Answer 12

Long patch repair is employed when a modified base resistant to AP lyase activity is present in DNA; it results in the replacement of ~2-10 nucleotides including the damaged base.

Question 13

What are the steps of Base Excision Repair (BER)?

Answer 13

1. DNA glycosylase recognizes a lesion in the DNA strand.
2. DNA glycosylase removes the damaged base, creating an AP site.
3. AP endonuclease (APE1) recognizes the AP site and creates a nick in the phosphodiester backbone.
4. The dRP is repaired by DNA pol β, LigIII, and XRCC1 (scaffold protein).

Question 14

What is an ‘AP site’ in DNA?

Answer 14

An apyrimidinic/apurinic (AP) site in DNA can be caused by base cleavage by DNA glycosylase or arise from spontaneous hydrolysis of the N-glycosidic bond.

Question 15

What are the steps of bacterial GG-NER?

Answer 15

1. The genome is interrogated by a UvrA₂:UvrB₂ heterotetramer. The UvrA homodimer is formed via its ABC ATPase module.
2. Once a lesion is recognized, UvrA₂ changes conformation, causing ATP to be hydrolyzed, which evicts UvrA₂.
3. The lesion is verified by UvrB helicase via its hairpin structure between the two strands.
4. UvrC is recruited, and it forms two incisions 12 nucleotides apart in the phosphodiester backbone of the damaged strand.
5. UvrD helicase unwinds the DNA, releasing the damaged strand which is repaired by DNA Pol I and ligase.

Question 16

Describe the steps of Eukaryotic GG-NER.

Answer 16

1. Lesion Recognition - The XPC complex recognizes a lesion by binding to bubble and loop structures (disrupted helix).
2. The TFIH helicase complex is recruited to the site. XPB and XPD open the DNA and verify the lesion due to stalling at sites with abnormal structure.
3. Lesion Processing - XPG and XPA bind to RPA, forming a large bubble that is cut by XPG and ERCC1-XPF. The lesion is repaired by a complex of RFC, PCNA, and Pol δ/ε.

Question 17

Describe the steps of Eukaryotic TC-NER.

Answer 17

1.

Question 18

Compare NHEJ and HRR

Answer 18

Both pathways repair DSB (double-stranded breaks)

NHEJ: Error-prone, occurs throughout the cell cycle (G1, S, G2), dependent on DNA-PK.

HRR: Not error-prone, active during S and G2, requires intact homologous template sequence (sister chromatid), dependent on ATM and BRCA.

Question 19

Describe the steps of NHEJ

Answer 19

1. DSB recognition by Ku70/80 binding to dsDNA breaks and recruiting DNA-PK to form a complex.
2. End processing dependent on the type of DSB. Ends are processed by a series of proteins ie MRN, CtIP, and Artemis. DNA-PK phosphorylates Artemis endonuclease, activating it to cleave the DNA to generate ends compatible for ligation.
3. Ligation occurs when DNA-PK/Ku complex recruits XRCC4 and Ligase IV, which ligate the DNA to repair the damage.

Question 20

What are the key steps in HRR?

Answer 20

1. Lesion recognition by ATM kinase and the MRN (MRE11, RAD50, and NBS1) complex is activated due to DSB. This process is suppressed by the Ku protein.
2. End resection by MRN to form ssDNA. May be dependent on BRCA1 and endonucleases.
3. Strand invasion and D-loop formation by RAD51. RPA ssDNA binding protein coats the resected DNA and acts as a substrate for RAD51, which forms holiday junctions with sister chromatids. The ssDNA is elongated by DNA Pol using the sister chromatid as a template.
4. Recombination and Ligation occur after the holiday junction is resolved.

Question 21

Which DNA repair pathway does the CRISPR system rely on for generating a gene knockout?

Answer 21

CRISPR knockouts rely on the error-prone NHEJ pathway.