Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III
Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III
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DOI：
10.1006/jmbi.2001.5382
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发表时间：
2002-02-22
影响因子：
5.6
通讯作者：
Wilson, DM
中科院分区：
文献类型：
--
作者：
Hadi, MZ;Ginalski, K;Wilson, DM
由于自发衰变、氧化或复制错误，非碱性位点和非常规3 '末端，例如3'-氧化片段（包括3 '-磷酸基团）和3'-错配核苷酸在基因组中以显著的频率出现。为了避免这些染色体修饰/中间体的潜在致突变或细胞毒性作用，生物体配备了脱嘌呤/脱嘧啶（AP）核酸内切酶和3 '-核酸酶。启动修复。Ape 1与大肠杆菌核酸外切酶M（ExoIII）具有同源性，是哺乳动物中主要的无碱基核酸内切酶，并且是3 '端加工的重要但选择性的贡献者。哺乳动物还具有与ExoIII具有序列同源性的第二种蛋白质（Ape 2），但该蛋白质表现出相对较弱的AP位点特异性和3 '-核酸酶活性。通过同源性建模研究证实，我们发现Ape 1的疏水口袋（由F266、W280和L282组成）中的取代使脱碱基切割效力降低约4倍至450，000倍，而将ExoIII样口袋引入Ape 2中增强了其A-P内切核酸酶功能。我们证明Ape 1的F266和W280突变增加了3'至5' DNA外切核酸酶活性。这些结果，再加上先前的比较序列分析，表明该活性位点疏水口袋影响具有保守的四层α/β折叠的序列相关蛋白质的不同集合的底物特异性。最后，我们报告野生型Ape 1切除3 '-错配核苷酸的速率比正确碱基配对的核苷酸高374倍，这在很大程度上取决于DNA底物的结构和序列，这表明人类蛋白质在3'-错配修复中具有新的选择性作用。(C)2002 Elsevier Science Ltd.
Abasic sites and non-conventional 3'-ends, e.g. 3'-oxidized fragments (including 3'-phosphate groups) and 3'-mismatched nucleotides, arise at significant frequency in the genome due to spontaneous decay, oxidation or replication errors. To avert the potentially mutagenic or cytotoxic effects of these chromosome modifications/intermediates, organisms are equipped with apurinic/apyrimidinic (AP) endonucleases and 3'-nucleases. that initiate repair. Ape1, which shares homology with Escherichia coli exonuclease M (ExoIII), is the major abasic endonuclease in mammals and an important, yet selective, contributor to 3'-end processing. Mammals also possess a second protein (Ape2) with sequence homology to ExoIII, but this protein exhibits comparatively weak AP site-specific and 3'-nuclease activities. Prompted by homology modeling studies, we found that substitutions in the hydrophobic pocket of Ape1 (comprised of F266, W280 and L282) reduce abasic incision potency about fourfold to 450,000-fold, while introduction of an ExoIII-like pocket into Ape2 enhances its A-P endonuclease function. We demonstrate that mutations at F266 and W280 of Ape1 increase 3' to 5' DNA exonuclease activity. These results, coupled with prior comparative sequence analysis, indicate that this active-site hydrophobic pocket influences the substrate specificity of a diverse set of sequence-related proteins possessing the conserved four-layered alpha/beta-fold. Lastly, we report that wild-type Ape1 excises 3'-mismatched nucleotides at a rate up to 374-fold higher than correctly base-paired nucleotides, depending greatly on the structure and sequence of the DNA substrate, suggesting a novel, selective role for the human protein in 3'-mismatch repair. (C) 2002 Elsevier Science Ltd.