Endonuclease V-mediated deoxyinosine excision repair in vitro
Endonuclease V-mediated deoxyinosine excision repair in vitro
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DOI：
10.1016/j.dnarep.2010.07.007
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发表时间：
2010-10-05
期刊：
影响因子：
3.8
通讯作者：
Fang, Woei-Horng
中科院分区：
文献类型：
--
作者：
Lee, Chia-Chia;Yang, Ya-Chien;Fang, Woei-Horng
DNA中的脱氧肌苷（dI）可以由脱氧腺苷的水解或亚硝化脱胺产生。它在大肠杆菌中由内切酶v （nfi基因产物）启动的修复途径中被切除。体外修复研究使用含有位点特异性脱氧肌苷的M13mp18衍生的异源双工，未配对的dI/G错配位于Xhol限制性内切酶的识别位点。我们的研究结果表明，在nfi（+）大肠杆菌提取物中，dI损伤得到了有效的修复，但在nfi突变体提取物中，修复水平大大降低。我们将含有脱氧肌苷的异双工置于由可溶性核酸内切酶V融合蛋白组成的纯化体系中。DNA聚合酶I和DNA连接酶，以及四种脱氧核苷三磷酸，有趣的是，我们发现这三种蛋白质单独足以有效地处理dI病变。我们还发现，DNA聚合酶I的3'-外切酶活性足以在这个最小的重构实验中去除dI病变(C) 2010 Elsevier B V所有权利保留
Deoxyinosine (dI) in DNA can arise from hydrolytic or nitrosative deamination of deoxyadenosine It is excised in a-repair pathway that is initiated by endonuclease V. the nfi gene product, in Escherichia col: Repair was studied in vitro using M13mp18 derived heteroduplexes containing a site-specific deoxyinosine Unpaired dI/G mismatch resides within the recognition site for Xhol restriction endonucleases, permitting evaluation of repair occurring on deoxyinosine-containing DNA strand Our results show that dI lesions were efficiently repaired in nfi(+) E coli extracts but the repair level was much reduced in nfi mutant extracts We subjected the deoxyinosine-containing heteroduplex to a purified system consisting of soluble endonuclease V fusion protein. DNA polymerase I. and DNA ligase, along with the four deoxynucleoside triphosphates Interestingly we found these three proteins alone are sufficient to process the dI lesion efficiently We also found that the 3'-exonuclease activity of DNA polymerase I is sufficient to remove the dI lesion in this minimum reconstituted assay (C) 2010 Elsevier B V All rights reserved