title: Defining amino acid residues involved in DNA-protein interactions and revelation of 3'-exonuclease activity in endonuclease V.
abstract: Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3' side one nucleotide from a deaminated base lesion. Site-directed mutagenesis analysis was conducted at seven conserved motifs of the thermostable Thermotoga maritima endonuclease V to probe for residues that affect DNA-protein interactions. Y80, G83, and L85 in motif III, H116 and G121 in motif IV, A138 in motif V, and S182 in motif VI affect binding of both the double-stranded inosine-containing DNA substrate and the nicked double-stranded inosine-containing DNA product, resulting in multiple enzymatic turnovers. The substantially reduced DNA cleavage activity observed in G113 in motif IV and G136 in motif V can be partly attributed to their defect in metal cofactor coordination. Alanine substitution at amino acid 118 primarily reduces the level of binding to the nicked product, suggesting that R118 plays a significant role in postcleavage DNA-protein interaction. Binding and cleavage analyses of multiple mutants at positions Y80 and H116 underscore the role these residues play in protein-DNA interaction and implicate their potential involvement as a hydrogen bond donor in recognition of deaminated DNA bases. DNA cleavage analysis using mutants defective in DNA binding reveals a novel 3'-exonuclease activity in endonuclease V. An alternative model is proposed that entails lesion specific cleavage and endonuclease to 3'-exonuclease mode switch by endonuclease V for removal of deaminated base lesions during endonuclease V-mediated repair.
authors.affiliation: Department of Genetics, Biochemistry and Life Science Studies, South Carolina Experiment Station, Clemson University, Room 219, Biosystems Research Complex, 51 New Cherry Street, Clemson, South Carolina 29634, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Feng
authors.forename: Liang
authors.initials: L
authors.lastname: Dong
authors.forename: Athena M
authors.initials: AM
authors.lastname: Klutz
authors.forename: Nima
authors.initials: N
authors.lastname: Aghaebrahim
authors.forename: Weiguo
authors.initials: W
authors.lastname: Cao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D004260:DNA Repair; D004722:Endoribonucleases / Q000737:chemistry* / Q000378:metabolism*; D005092:Exonucleases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D012568:Schizosaccharomyces / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases; D005092:Exonucleases; C022650:spleen exonuclease; C025115:ribonuclease V