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NMR Studies of the DNA Polymerase β L22P Variant
Author(s) -
KIRBY THOMAS
Publication year - 2013
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.27.1_supplement.lb77
Subject(s) - dna polymerase , chemistry , polymerase , methionine , dna , lyase , microbiology and biotechnology , heteronuclear single quantum coherence spectroscopy , amino acid , stereochemistry , enzyme , biochemistry , biology , nuclear magnetic resonance spectroscopy
The gastric cancer‐associated DNA Polymerase β (Polβ) variant with Lys22 replaced by Pro (L22P), has been shown to exhibit reduced DNA binding as well as reduced DNA lyase activity, and to be deficient in base excision repair [Dalal,S., Chikova,A., Jaeger,J., and Sweasy,J.B. (2008) Nucleic Acids Res., 36, 411–422]. The amino acid change is in the middle of helix1, the first of 4 helices that make up the Polβ lyase domain. We have used NMR experiments with labeled L22P to probe the structure of the variant in solution. The 1 H‐ 13 C HSQC resonances of 13 C methyl methionine‐labeled enzyme indicate that the L22P mutation appears to structurally disrupt at least part of the lyase domain, as judged by the chemical shift and intensity of the M18 resonance. The rest of the methionine methyl resonances, which arise from the polymerase domain, are unaffected by the mutation. The methionine methyl resonances of wild‐type Polβ exhibit characteristic perturbations upon formation of binary and ternary complexes with gapped DNA and a complementary dNTP. Under the same conditions, the methionine resonances of L22P show none of the changes associated with complex formation. Consistent with these observations, the 1 H‐ 15 N HSQC spectrum of the isolated 8K lyase domain of the L22P Polβ variant is characteristic of a disordered protein.