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Specificity and Activity of DNA Polymerase DinB and Polκ Loop Swap Variants
Author(s) -
Packer Morgan Renee,
Antczak Nicole,
Beuning Penny
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1052.2
Subject(s) - dna polymerase , processivity , biology , genetics , dna polymerase ii , polymerase , dna , microbiology and biotechnology , reverse transcriptase , gene , polymerase chain reaction
DNA damage is ubiquitous, arising from both natural and artificial sources. The presence of damaged bases in DNA requires the use of specialized Y‐family DNA polymerases, which have the ability to catalyze the insertion of a nucleotide opposite a damaged base and to continue elongation past this DNA lesion. This process of DNA replication past damaged nucleotide(s) is known as translesion synthesis. A major question is how different Y‐family DNA polymerases achieve specificity for different types of DNA damage. Previous work showed that the minor groove N 2 ‐furfuryl‐deoxyguanosine lesion is the cognate lesion for both E. coli DinB and its human ortholog Polκ and that both DinB and Polκ are inhibited by bulky major groove lesions. It was further determined that the DinB loop1 R35A mutation enables the protein to bypass the non‐cognate N 6 ‐furfuryl‐deoxyadenosine major groove lesion (Walsh, et al. 2013 DNA Repair 12:713). Our research probes the contributions of the loop1 region to the specificity and activity of DinB and Polκ, which is being assessed via the analysis of several loop swap variants that represent step‐wise progressive changes from the native loop1 to that of DinB, Polκ, or Dpo4. Dpo4 is of interest as it is a relatively non‐specific Y‐family polymerase. We show that DinB and Polκ have high specificity for their cognate lesion, and Polκ variants containing the DinB loop1 sequence retain activity similar to wild‐type Polκ, whereas the DinB variants do not. Thus, Polκ is more resistant to changes in its amino acid sequence. Support or Funding Information This work was funded by a National Science Foundation Research Experience for Undergraduates grant and the Department of Chemistry and Chemical Biology at Northeastern University.