A bacterium E. coli suffered ultraviolet irradiation. In the base-pair sequence

Question

A bacterium E. coli suffered ultraviolet irradiation. In the base-pair sequence shown below, note that the adjacent thymines formed a dimer. What are the requirements, the process, and the outcome of the repair by (a) photoreactivation and (b) general excision repair, if this damage were to be repaired? PCR (polymerase chain reaction) was used to amplify a small region at the 5' end of the coding region of three mutant alleles in yeast. This region was sequenced as follows, grouped in codons: Classify these mutations into as many categories as possible. Mutant 1 Mutant 2 Mutant 3

Explanation / Answer

5. (a) Photoreactivation:

Requirement- DNA photolyases

Process- Kink in the DNA is recognized and bound by DNA photolyases. When blue light is used for exciting, the conformation is changed by photolyases which breaks apart the dimer.

Outcome- Thymine dimers will be broken down and repaired.

(b) General excision repair-

Requirement- (A)BC exinuclease

Process- Damage in the DNA is recognized by UvrA. After detection UvrB is lead to lesion by UvrA. UvrB proceeds to melt the DNA and UvrA detaches from the UvrB-DNA complex. After this UvrC attaches to the UvrB-DNA complex. 3’ and 5' incision made by UvrB and UvrC respectively. This cleavage results in the excision of area of the bubble which includes the lesion. The short, single-stranded DNA segment that is created is then removed by a DNA helicase, UvrD. Undamaged, complementary strand is used by DNA polymerase as a template to replace the excised DNA. Finally DNA ligase causes the formation of covalent phosphodiester bonds between the 3’ hydroxyl end of one nucleotide and 5’ phosphate end of another nucleotide.

Outcome- Original nucleotide sequence would be restored.

Note- This damage can be repaired by photoreactivation.

6. (a) Missense mutation since AAT changed to ATT which changed the amino acid being coded.

(b) Silent mutation because AAT changed to AAG which caused the same amino acid to be coded.

(c) Insertion because T is added before AAT.

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