DNA Damage and Repair

Question 1

Mutagens

Answer 1

Chemical agents that alter DNA bases

Question 2

DNA bases can be altered through:

Answer 2

• Oxidation
• Deamination
• Alkylation
• UV radiation
• X-ray exposure

Question 3

Guanine Oxidation

Answer 3

Free O radicals produced

G-A bp instead of G-C results in point mutation after replication

Question 4

Cytosine Deamination

Answer 4

• Occurs 100x/day in mammalian cell
• Deamination: loss of exocyclic amino group
• Cytosine deamination is spontaneous in cell, cytosine reacts with water, forms uracil + NH3

Question 5

Adenine Deamination

Answer 5

Occurs spontaneously in the cell

• Adenine + H2O -> hypoxanthine + NH3
• Results in C-A bp

Question 6

5-Methylcytosine Demination

Answer 6

5-Methylcytosine + H2O -> thymine + NH3

Cytosine is methylated to control gene expression

Question 7

Guanine Alkylation

Answer 7

Alkylation: addition of hydrocarbon
- Aflatoxin B1

Aflatoxin B1 -> Alfatoxin B1 epoxide (activated) -(reacts with DNA)-> deoxyribose attached to DNA adduct (alkylated guanine)

Distorts the structure of DNA and messes up replication via stalling polymerase or addition of the wrong dNTP

Question 8

Thymine Dimer (UV Damage)

Answer 8

UV covalently links adjacent pyrimidines along the DNA strand = structure distortion

Question 9

DNA Repair

Answer 9

1. Damage and repair are occurring constantly
2. Often, repair will restore the genetic info
3. Sometimes it is not possible to restore the original info, so the cell may use approx repairs and/or undergo apoptosis

Question 10

Steps for repair are:

Answer 10

1) Recognition of damage
2) Remove defective base/region
3) Repair with correct sequence

Question 11

Mismatch repair

Answer 11

• Mismatch is recognized by MutS
• MutL binds and recruits MutH (endonuclease)
• Exonuclease excises incorrect region
• DNA pol III fills the gap

Question 12

How does the repair machinery know which DNA strand has the correct base?

Answer 12

Template/parent strand is methylated and the new strand is unmethylated

Question 13

Direct Repair

Answer 13

• Repair without removing fragments of DNA
• Photochemical cleavage of pyrimidine dimers by DNA photolyase
• Uses energy of visible light to break cyclobutane ring

Question 14

Nucleotide Excision Repair (thymine dimers in the dark)

Answer 14

• Recognizes distortions in helix
• Uvr ABC (excinuclease) cuts DNA @ 2 sites
• DNA pol I fills the gap
• DNA ligase repairs the phosphodiester backbone

Question 15

Base Excision Repair

Answer 15

1) Defective base flipped into glycosylase active site = glucosidic bond cleavage by uracil glycosylase
2) AP endonuclease nicks phosphodiester backbone
3) Deoxyribose phosphodiesterase removes deoxyribose phosphate unit
4) DNA pol I inserts correct nucleotide
5) DNA ligase seals gap

Question 16

DNA typically uses thymine instead of uracil, which is found in RNA. Why?

Answer 16

Thymine instead of Uracil allows the cell to distinguish between deamination of cytosine forming uracil and the correct base

Question 17

DNA Recombination

Answer 17

In genetic recombination, 2 daughter molecules of DNA are formed by the exchange of genetic material between 2 parent molecules
- Recombination can be used to repair double-strand breaks

Question 18

Repair of Double-Strand Break Using Recombination

Answer 18

1) 5’exonuclease generates ssDNA at the site of the break
2) Strand invasion and formation of a displacement loop
3) DNA synthesis
4) Second strand invasion resulting in the formation of 2 holliday junctions
5) Holliday junctions are cleaved and ligated