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Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues.
Robey-Bond, Susan M; Benson, Meredith A; Barrantes-Reynolds, Ramiro; Bond, Jeffrey P; Wallace, Susan S.
Afiliación
  • Robey-Bond SM; Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, United States.
  • Benson MA; Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, United States.
  • Barrantes-Reynolds R; Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, United States.
  • Bond JP; Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, United States.
  • Wallace SS; Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, United States. Electronic address: swallace@uvm.edu.
DNA Repair (Amst) ; 53: 43-51, 2017 05.
Article en En | MEDLINE | ID: mdl-28292631
The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar kcat/Kd values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Daño del ADN / Dominio Catalítico / Proteínas de Escherichia coli / Desoxirribonucleasa (Dímero de Pirimidina) / Mutación Límite: Humans Idioma: En Revista: DNA Repair (Amst) Asunto de la revista: BIOLOGIA MOLECULAR / BIOQUIMICA Año: 2017 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Daño del ADN / Dominio Catalítico / Proteínas de Escherichia coli / Desoxirribonucleasa (Dímero de Pirimidina) / Mutación Límite: Humans Idioma: En Revista: DNA Repair (Amst) Asunto de la revista: BIOLOGIA MOLECULAR / BIOQUIMICA Año: 2017 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Países Bajos