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7.E : Mutation and Repair of DNA (Exercises)

Q7.12

If the top strand of the segment of DNA GGTCGTT were targeted for reaction with nitrous acid, and then it underwent two rounds of replication, what are the likely products?

Q7.13

Are the following statements about nucleotide excision repair in E. coli true or false?

  1. UvrA and UvrB recognize structural distortions resulting from damage in the DNA helix.
  2. In a complex with UvrB, UvrC cleaves the damaged strand on each side of the lesion.
  3. The helicase UvrD unwinds the DNA, thereby dissociating the damaged patch.

Q7.14

Are the following statements about mismatch repair in E. coli true or false?

  1. MutS will recognize a mismatch.
  2. MutL, in a complex with ATP, will bind to the MutS (bound to the mismatched region) and activate MutH.
  3. MutH will cleave 5' to the G of the nearest methylated GATC motif (GmeATC).
  4. The mismatch repair system can discriminate between old versus newly synthesized strands of DNA.

For the next 6 problems, consider the following DNA sequence, from the first exon of the HRAS gene. A transversion of G to T at position 24 confers anchorage independence and tumorigenicity to NIH 3T3 cells (fibroblasts). This mutation is one step in tumorigenic transformation of bladder cells, and it likely plays a role in other cancers.

 

            10         20         30

5'  TAAGCTGGTG GTGGTGGGCG CCGGCGGTGT

3'  ATTCGACCAC CACCACCCGC GGCCGCCACA

Q7.15

hat would the sequence be if the G at position 14 (top strand) were alkylated at the O6 position by MNNG and then went through 2 rounds of replication?

Q7.16

hat would the sequence be if the C at position 24 (bottom strand) were oxidized by HNO2 and then went through 2 rounds of replication?

Q7.17

What would happen if this sequence were irradiated with UV at a wavelength of 260 nm?

Q7.18

If you were in charge of maintaining this DNA sequence, and you had the enzymatic tools known in E. coli, how would you repair the damage from question 7.15? Consider what would happen if the damage were corrected before or after replication. 

Q7.19

How could

  1. the oxidative damage in problem 7.16 or
  2. the UV products in problem 7.17 be repaired?

Q7.20

Let's say that a C to A transversion occurred at position 24 on the bottom strand of the segment below, and that a segment with a GATC is located about 300 bp away.

            10         20         30 ...      m

5'  TAAGCTGGTG GTGGTGGGCG CCGGCGGTGT ...GGACGGATCC

3'  ATTCGACCAC CACCACCCGC GGCAGCCACA ...CCTGCCTAGG

If this DNA is marked by the dam methylase system similarly to E. coli, how would the mismatch at position 24 be repaired? How does the cell decide which is the correct nucleotide, and what enzymes would be used? Explain how the enzymes work in this specific example.

Q7.21

The following is paraphrased from a presentation at the year 2000 meeting of the American Society for Human Genetics .

Fanconi anemia (FA) is an autosomal recessive disease associated with cancer predisposition. Cultured cells from FA patients have high levels of spontaneous chromosome breaks, suggesting that FA cells may have a defect in DNA repair. To test this hypothesis, DNA end-joining activity was measured in nuclear extracts from diploid fibroblasts belonging to FA complementation groups A and D, and from several normal donors. Extracts from normal donors (controls) efficiently joined linear plasmid substrates, but extracts from FA fibroblasts had only 10% the activity of the normal controls. Addition of FA extract to normal cell extract had no effect on the activity of the latter. However, when extracts from fibroblasts of FA complementation group A were combined with those of complementation group D, normal levels of DNA end-joining activity were reconstituted.

What do you conclude from these data?

Q7.22

How would you use dut, ung mutants to select for site-directed mutations?