Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Nucleic Acids Res. 2010 Apr;38(7):2291-301. doi: 10.1093/nar/gkp1210. Epub 2010 Jan 7.
Uracil DNA glycosylase (Ung) initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by approximately 7- and approximately 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed approximately 5-, approximately 3.0- and approximately 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.
尿嘧啶 DNA 糖基化酶 (Ung) 启动尿嘧啶切除修复途径。我们之前已经对 Ung 的 Y66W 和 Y66H 突变体进行了表征，结果表明它们的尿嘧啶切除活性分别降低了约 7 倍和约 170 倍。在这项研究中，荧光各向异性测量表明，与野生型相比，Y66W 蛋白在与 AP-DNA 的结合中中度受损和减弱。用 ung::kan、ungY66H:amp 或 ungY66W:amp 等位基因替代大肠杆菌中的 ung，突变频率分别增加了约 5 倍、约 3.0 倍和约 2.0 倍。对 rpoB 中利福平抗性决定区的突变分析表明，Y66W 等位基因导致 A 到 G（或 T 到 C）突变增加。然而，在质粒携带基因表达野生型 Ung 时，A 到 G 突变的增加得到了缓解。生化和计算分析表明，Y66W 突变体仍然严格特异性地从 DNA 中切除尿嘧啶。有趣的是，缺乏 AP 内切酶的菌株也显示出 A 到 G 突变的增加。我们在这样一个提议的背景下讨论这些发现，即 DNA 糖基化酶在它们产生的 AP 位点上的停留时间会保护它们，直到招募 AP 内切酶进行进一步修复。
