DNA damage and repair

Study questions.

1. Which of the following statements about DNA polymerase I are correct?

a) It adds deoxyribonucleotide units to the 3'-hydroxyl of a primer.

b) It uses the template strand to select the deoxyribonucleotide unit to add to the growing DNA chain.

c) It contains a 3' Õ 5' nuclease that cleaves phosphodiester bonds to yield 3'-dNMPs and 3'-phosphate-terminated DNA. yield 3'-OH ends.

d) It contains two nuclease activities in the same polypeptide chain that contains the polymerase active site.

e) It can be cleaved with a protease into two fragments, each of which has a nuclease activity.

ANSWER: A, B, D and E

COMMENTS: Unlike DNA pol I, DNA pol III does not have a 5'-exonuclease and so, it cannot remove RNA primers from Okazaki fragments. However, pol III is more "processive" than pol I and can interact with other proteins necessary for replication, like the primase-helicase complex.

2. What property of DNA allows the repair of some residues damaged through the action of mutagens? Complementarity of the two strands allows for information to be recovered through using the undamaged strand as template.

3. How does the repair machinery of E. coli identify a DNA strand that has recently misincorporated a noncomplementary nucleotide during replication in order to repair it? Through the methyl-directed mismatch repair pathway.

4. Which of the following enzymes or processes can be involved in repairing DNA in E. coli damaged by uv light-induced formation of a thymine dimer?

a) DNA ligase seals the newly synthesized strand to undamaged DNA to form the intact molecule.

b) The UvrABC enzyme (excinuclease) hydrolyzes phosphodiester bonds on both sides of the thymine dimer.

c) DNA polymerase I fills in the gap created by the removal of the oligonucleotide bearing the thymine dimer.

d) The UvrABC enzyme recognizes a distortion in the DNA helix caused by the thymine dimer.

e) A photoreactivating enzyme absorbs light and cleaves the thymine dimer to re-form two adjacent thymine residues.

ANSWER: All are correct.

COMMENTS: I did not discuss photoreactivation in the lectures. This enzyme system, which is highly efficient in bacteria, appears to be absent in humans, except perhaps for some lymphocytes. Presence of efficient DNA repair in lymphocytes may help diminish harmful effects on the immune system by the Sun's UV-A rays (325-400nM), which penetrate deeper into the skin layers than do UV-B and UV-C rays (<325nM).

5. Humans suffering from xeroderma pigmentosum develop skin cancers when exposed to sunlight because they have a deficiency in:

a) RNA primase.

b) DNA recombinase.

c) Uracil-N-glycosylase.

d) an enzyme of the excision repair pathway.

e) an enzyme of the mismatch-repair pathway.

ANSWER: D

COMMENTS: Refer to Dr. Hamori's lectures.

6. If cytosine in DNA is deaminated, the resulting base is:

a) removed by an endonuclease.

b) removed by an exonuclease.

c) removed by a glycosylase.

d) not removed, but ultimately replaced by cytosine in subsequent DNA replication.

e) not recognized by DNA polymerase, and thus inhibits DNA replication.

ANSWER: C; specifically the Uracil-N-glycosylase.

 

7. Eukaryotic DNA is highly methylated at the C-5 position of cytosine. The degree of methylation is inversely correlated with gene expression. Although the exact role of C-5 methylation in gene expression is not known, it is known that these C-5-methylated cytosines are a source of mutations. Explain why

ANSWER: Because when 5-methylcytosine is oxidatively deaminated it becomes "thymine", which is a normal DNA base and is not removed by glycosylases. It ultimately leads to transition (C to T) mutations (Me-C:G® T:G ® T:A & C:G)

8. Explain the principles of error-prone DNA repair.

ANSWER: Error-prone repair happens when "untemplated" DNA synthesis proceeds over a modified or an abasic (lost base) nucleotide position in the DNA. Two-thirds of the time, the mutations are transversions.