Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues

• Published in: DNA repair Vol. 53; pp. 43 - 51
• Main Authors: Robey-Bond, Susan M., Benson, Meredith A., Barrantes-Reynolds, Ramiro, Bond, Jeffrey P., Wallace, Susan S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-05-2017
• Subjects: Amino Acid Sequence; Base excision repair; Catalytic Domain; Deoxyribonuclease (Pyrimidine Dimer) - genetics; Deoxyribonuclease (Pyrimidine Dimer) - metabolism; DNA - metabolism; DNA Damage; Endonuclease III; Enzyme kinetics; Escherichia coli - enzymology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Humans; Kinetics; Mutation; Substrate Specificity; DHU; DHT; Endonuclease III; Tg; EcoNth; DTT; hNTHL1; Enzyme kinetics; Base excision repair
• ISSN: 1568-7864; 1568-7856
• DOI: 10.1016/j.dnarep.2017.02.014

•E. coli Asp44 plays a different role than its homologous residue in human NTHL1.•hNTHL1 Gln287 in involved in opposite base specificity as well as catalysis.•EcoNth Thr121 is involved in DNA binding and catalysis.•EcoNth and hNTHL1 demonstrate similar substrate specificity for DHT.
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.