Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.
Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases.
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DOI：
10.1074/jbc.m112.367243
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发表时间：
2012-07-27
期刊：
影响因子：
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通讯作者：
McHugh PJ
中科院分区：
文献类型：
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作者：
Sengerová B;Allerston CK;Abu M;Lee SY;Hartley J;Kiakos K;Schofield CJ;Hartley JA;Gileadi O;McHugh PJ
背景：核酸酶hSNM1A和hSNM1B参与DNA链间交联修复。结果：hSNM1A和hSNM1B采用未损伤和交联DNA进行生化表征。建立了一种适合于抑制剂鉴定的核酸酶实时检测方法。结论：高分子量和交联DNA优先激活hSNM1A。意义：本工作为hSNM1A抑制剂的开发改善肿瘤治疗提供了基础。细胞研究表明，人类SNM1A和SNM1B/Apollo都与DNA链间交联（ICLs）的修复有关，并且SNM1B也是端粒保护所必需的。在这里，我们描述了SNM1A和SNM1B蛋白的生化特性的研究。结果揭示了这两种蛋白质在机制上的一些根本差异。SNM1A和SNM1B在二价阳离子刺激下以5′- 3′的方向消化双链和单链DNA，两种核酸酶都被锌螯合剂邻菲罗啉抑制。我们发现SNM1A对单链DNA比双链DNA具有更大的亲和力，而SNM1B没有观察到这一点。尽管这两种蛋白在低分子量DNA寡核苷酸底物上表现出低水平的加工能力，但当呈现高分子量DNA时，SNM1A单独呈现出更高的活性，能够消化数千碱基长的DNA。这两种蛋白都可以消化由非扭曲小槽交联剂SJG-136诱导的过去的icl，尽管SNM1A表现出更大的能力。这与SNM1A和SNM1B可能在ICL修复中表现出一些冗余的建议是一致的。总之，我们的工作确定了SNM1A和SNM1B在底物选择性上的差异，这可能与它们在体内的作用有关，并且可能在选择性抑制剂的开发中被利用。
Background: The nucleases hSNM1A and hSNM1B are implicated in DNA interstrand cross-link repair. Results: hSNM1A and hSNM1B were biochemically characterized using undamaged and cross-linked DNA. A real-time assay for the nucleases suitable for inhibitor identification was developed. Conclusion: Preferential hSNM1A activation by high molecular weight and cross-linked DNA was observed. Significance: This work provides a basis for hSNM1A inhibitor development for improved cancer therapy. Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5′-to-3′ directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.