title: The carboxyl terminal of the archaeal nuclease NurA is involved in the interaction with single-stranded DNA-binding protein and dimer formation.
abstract: The nuclease NurA is present in all known thermophilic archaea and has been implicated to facilitate efficient DNA double-strand break end processing in Mre11/Rad50-mediated homologous recombinational repair. To understand the structural and functional relationship of this enzyme, we constructed five site-directed mutants of NurA from Sulfolobus tokodaii (StoNurA), D56A, E114A, D131A, Y291A, and H299A, at the conserved motifs, and four terminal deletion mutants, StoNurAΔN (19-331), StoNurAΔNΔC (19-303), StoNurAΔC (1-281), and StoNurAΔC (1-303), and characterized the proteins biochemically. We found that mutation at the acidic residue, D56, E114, D131, or at the basic residue, H299, abolishes the nuclease activity, while mutation at the aromatic residue Y291 only impairs the activity. Interestingly, by chemical cross-linking assay, we found that the mutant Y291A is unable to form stable dimer. Additionally, we demonstrated that deletion of the C-terminal amino acid residues 304-331 of StoNurA results in loss of the physical and functional interaction with the single-stranded DNA-binding protein (StoSSB). These results established that the C-terminal conserved aromatic residue Y291 is involved in dimer formation and the C-terminal residues 304-331 of NurA are involved in the interaction with single-stranded DNA-binding protein.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan 250100, People's Republic of China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wei
authors.forename: Songtao
authors.initials: S
authors.lastname: Zhang
authors.forename: Linlin
authors.initials: L
authors.lastname: Hou
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Ni
authors.forename: Duohong
authors.initials: D
authors.lastname: Sheng
authors.forename: Yulong
authors.initials: Y
authors.lastname: Shen
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000235:genetics; D019281:Dimerization; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D005557:Formaldehyde / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D016944:Sulfolobus / Q000201:enzymology
chemical_list: D019843:Archaeal Proteins; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D005557:Formaldehyde; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases