The carboxyl terminal of the archaeal nuclease NurA is involved in the interaction with single-stranded DNA-binding protein and dimer formation
The carboxyl terminal of the archaeal nuclease NurA is involved in the interaction with single-stranded DNA-binding protein and dimer formation
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DOI：
10.1007/s00792-010-0351-2
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发表时间：
2010
期刊：
影响因子：
2.9
通讯作者：
Tao Wei;Song-tao Zhang;Linlin Hou;J. Ni;Duohong Sheng;Yulong Shen
中科院分区：
文献类型：
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作者：
Tao Wei;Song-tao Zhang;Linlin Hou;J. Ni;Duohong Sheng;Yulong Shen
核酸酶NurA存在于所有已知的嗜热古菌中，并被认为在Mre11/ rad50介导的同源重组修复中促进DNA双链断裂端高效加工。为了了解该酶的结构和功能关系，我们从sulfolobus tokodaii（StoNurA）中构建了5个位点导向的NurA突变体，分别是D56A、E114A、D131A、Y291A和H299A，在保守基序上，以及4个末端缺失突变体StoNurAΔN（19-331）、StoNurAΔNΔC（19-303）、StoNurAΔC（1-281）和StoNurAΔC(1-303)，并对这些蛋白进行了生化表征。我们发现，酸性残基D56、E114、D131和碱性残基h99的突变会使核酸酶活性消失，而芳香残基Y291的突变只会使酶活性受损。有趣的是，通过化学交联实验，我们发现突变体Y291A无法形成稳定的二聚体。此外，我们还证明了StoNurA c端氨基酸残基304-331的缺失导致其与单链dna结合蛋白（StoSSB）的物理和功能相互作用的丧失。这些结果表明，c端保守芳香残基Y291参与了二聚体的形成，而NurA的c端残基304-331参与了与单链dna结合蛋白的相互作用。
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 fromSulfolobus 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.