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Human Y‐family DNA polymerase kappa is more tolerant of amino acid substitutions in the active site loop region than its E. coli ortholog DinB
Author(s) -
Antczak Nicole M,
Ippoliti Paul,
Walsh Jason,
Packer Morgan,
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.1052.1
Subject(s) - dna polymerase , biology , dna polymerase ii , polymerase , primer (cosmetics) , mutagenesis , dna clamp , dna , genetics , microbiology and biotechnology , biochemistry , mutation , chemistry , gene , polymerase chain reaction , reverse transcriptase , organic chemistry
More than 100,000 lesions are generated on DNA in each cell per day due to such agents as reactive oxygen species and ultraviolet radiation. Y‐family DNA polymerases are specialized to carry out translesion synthesis by replicating damaged DNA and are conserved in all domains of life. There are two Y‐family polymerases in E. coli , DinB (Pol IV) and UmuD′ 2 C (Pol V), one in the archaeon Sulfolobus solfataricus , Dpo4, as well as several in humans including Pol κ (hPol κ), which is a DinB ortholog. Both DinB and hPol κ are known to bypass minor groove adducts of guanine whereas Dpo4 can bypass both minor and major groove adducts. Using primer extension assays on DNA substrates containing the minor groove adduct N 2 ‐furfuryl‐dG as well as undamaged templates combined with site‐directed mutagenesis to construct polymerase variants, we found that loop 1 residues of DinB near the catalytic site are important for activity as well as the selection of the correct nucleotide. There is a similar trend in the loop 1 region of hPol κ. In single nucleotide incorporation assays, wild‐type and variants of both polymerases show a tendency to insert incorrect dT as well as the correct dC opposite the N 2 ‐furfuryl‐dG adduct. This is also seen for wild‐type and variants of hPol κ on an undamaged DNA template. These observations suggest that the loop 1 region of both polymerases is important in correct nucleotide incorporation during translesion synthesis. Furthermore, although both DinB and hPol κ are inhibited by major groove modifications, hPol κ is more active than DinB on DNA with major groove adducts. In order to probe this further, we used primer extension assays with DNA substrates containing the minor groove adduct N 2 ‐furfuryl‐dG, major groove adduct N 6 ‐furfuryl‐dA, as well as undamaged templates, with loop swap constructs of DinB and hPol κ, we found the loop regions of DinB and hPol κ near the catalytic site are important for bypass of different types of DNA damage and insertion of the correct nucleotide. Specifically, hPol κ is more tolerant of substitutions in its active site loops than DinB is. By creating loop swaps, an increase in the understanding of the preferential bypass of minor groove adducts by DinB and hPol κ can be accomplished. Support or Funding Information Support from American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.