Rothwell D G, Hickson I D
Imperial Cancer Research Fund Laboratories, University of Oxford, John Radcliffe Hospital, UK.
Nucleic Acids Res. 1996 Nov 1;24(21):4217-21. doi: 10.1093/nar/24.21.4217.
HAP1 is a divalent cation-dependent endonuclease from human cells with specificity for apurinic/apyrimidinic (AP) sites in DNA. Extraction of the essential metal ion from purified HAP1 stabilized its binding to an oligonucleotide containing a single AP site, permitting AP site binding studies to be undertaken using gel retardation assays. Binding of HAP1 to such an oligonucleotide was dependent upon the presence of an AP site. Previous structural and modelling studies have suggested a role for Asn212 (Asn153 in exonuclease III, the bacterial homologue of HAP1) in substrate recognition. Substitution of alanine for Asn212 abolished the AP endonuclease activity of purified recombinant HAP1 protein. More conservative substitutions of aspartate or glutamine for Asn212 still led to a reduction in specific activity of at least 300-fold. Moreover, none of the three Asn212 substitution mutants of HAP1 possessed detectable AP site binding activity in vitro. This study indicates that chelation of the active site metal ion in HAP1 stabilizes the complex of the protein with AP sites and identifies an active site asparagine residue as an important component of AP site recognition by the HAP1 protein.
HAP1是一种来自人类细胞的二价阳离子依赖性核酸内切酶，对DNA中的无嘌呤/无嘧啶（AP）位点具有特异性。从纯化的HAP1中提取必需金属离子可稳定其与含有单个AP位点的寡核苷酸的结合，从而能够使用凝胶阻滞试验进行AP位点结合研究。HAP1与这种寡核苷酸的结合取决于AP位点的存在。先前的结构和建模研究表明，Asn212（在HAP1的细菌同源物核酸外切酶III中为Asn153）在底物识别中起作用。用丙氨酸替代Asn212消除了纯化的重组HAP1蛋白的AP核酸内切酶活性。用天冬氨酸或谷氨酰胺对Asn212进行更保守的替代仍导致比活性至少降低300倍。此外，HAP1的三个Asn212替代突变体在体外均不具有可检测的AP位点结合活性。这项研究表明，HAP1活性位点金属离子的螯合稳定了蛋白质与AP位点的复合物，并确定活性位点天冬酰胺残基是HAP1蛋白识别AP位点的重要组成部分。
