DNA Mutation/Repair

Question 1

Enhancement of Replication Fidelity

Answer 1

1. Low error rat of DNA Pol (1 in 10,000)
2. 3’ exonuclease activcity (1 in 1,000)
3. Repair systems (mismatch repairs) lowers error rate an addition 1000 fold

Question 2

Mis-incorporation of nucleotides during DNA replication

Answer 2

Adenine tautomer can base pair Guanine

Question 3

Inherent chemical instability of baes

Answer 3

1. Deamination of C–>U
2. Hydrolysis of N-glycosidic bond - depurination

Question 4

Environmental mutagens

Answer 4

Chemical mutagens - deaminating agents (C->U; G–> X)

Deamination caused by nitrous acid precursors

Alkylating agents (often N7 of guanine)

• Alkylating agents: nitrogen mustards, aflatoxin, bezo(a)pyrene
  - When guanine is methylated to O6-methylguanine it can pair with thymine rather than cytosine

Question 5

Ionizing radiation - UV light

Answer 5

Can cause pyrimidine dimers, photolyase repair

Question 6

Intercalating agents

Answer 6

Lead to insertion or deletion of one or more basepairs, alters reading frames; frameshift mutations caused by flat aromatic molecules

Question 7

Multiple DNA Repair Systems

Answer 7

1. Mismatch repair - mismatches
2. Base-excision repair - removes abnormal/damaged bases
3. Nucleotide-excision repair - removes large structural damages (thymine dimers)
4. Direct repair - system that recognize and repair with specific damages
5. Recombination repair - uses sister chromatid to repair damaged region

Question 8

DNA Methylation and Mismatch Repair

Answer 8

DNA methylation tags undamaged DNA. Thymine used in DNA instead of uracil to enhance the fidelity of the genome

Tags distinguishes parental DNA from newly synthesized daughter strand

Question 9

Methyl directed mismatch repair

Answer 9

Recognition of the seuence 5’GATC and of the mismatch are specialized functions of the MutH and MutS proteins, respectively.

The Mut L proteins form a complex with MutS at the mismatch,

DNA is threaded through this complex such that the complex moves simultaneously in both directions along the DNA until encounter a MutH protein bound at a hemimethylated GATC sequence.

MutH cleaves the unmethylated strand on the 5’ side of the G in this sequence.

A complex consisting of DNA helicase II and one of the several exonucleases then degraes the unmethylated DNA strand from that point toward the mismatch

Question 10

Base-Excision Repair

Answer 10

1. Deamination of cytosine leads to uracil which DNA glycosylase recognizes as foreign and hydrolyzes the glycosidic bond
2. An AP endonuclease cleaves the phosphodiester backbone near the AP site
3. DNA pol I initiates repair synthesis from the free 3’ hydroxyl at the nick
4. The nick is sealed by DNA ligase

Question 11

Nucleotide Excision Repair

Answer 11

Removes large structural damages

1. An excinuclease binds to DNA at the site of a bulky lesion and cleaves the damaged DNA strand on either side of the lesion
2. The DNA segment is removed with the aid of a helicase
3. The gap is filled in by DNA polymerase I
4. The remaining nick is sealed with DNA ligase

Question 12

Direct Repair

Answer 12

Some forms of DNA damage can be enzymaticaly reversed.

1. Removal of alkyl groups at the O 6 posiiton of guanine by MGMT

Inhibition of MGMT can faciliate cancer chemo b/c cancer cells can upregualte this enzyme in response to chemotherapeutic agents

Question 13

Recombination Repair

Answer 13

Certain types of lesions in DNA, such as double-stranded breaks, double strand cross-links, and lesions in single stranded regions cannot be repaired using information from the complimentary strand.

Information must come from a separate homologous chromosome.

Question 14

Xeroderma Pigmentosum (XP)

Answer 14

A rare skin disease; individiuals are extremely sensitive to UV light and develop sign cancer which metatasize

Results in a defect in the nucleotide excision reapir mechanism for thymine dimers

Question 15

Breast Cancer

Answer 15

Some forms of breast cancer are due to defects in breast cancer susceptibility genes (BRCA). BRCA genes participate in DNA repair

Question 16

Major mechanisms of DNA damage

Answer 16

Spontaneous and radiation-based mechanisms. Can be silient (non aa chance), result in mis-sense (aa change), nonsense (stop codon insertions), or frameshift (insertions and deletions) mutations

Question 17

Mechanisms of DNA repair

Answer 17

1. Mismatch repair
2. Base excision repair
3. Nucleotide excision repair
4. Direct repair
5. Recombinational repair

Question 18

Defects in DNA damage repair and disease

Answer 18

• Mutations or inherited defects in DNA repair pathways lead to increased sensitivty to DNA damaging agents
• Most often manifestd in inreased cancer rates
• Most deleterious damage involves double strand DNA breaks