The dCMP transferase activity of yeast Rev1 is biologically relevant during the bypass of endogenously generated AP sites.
The dCMP transferase activity of yeast Rev1 is biologically relevant during the bypass of endogenously generated AP sites.
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DOI：
10.1016/j.dnarep.2011.09.017
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发表时间：
2011-12-10
期刊：
影响因子：
3.8
通讯作者：
Jinks-Robertson S
中科院分区：
文献类型：
--
作者：
Kim N;Mudrak SV;Jinks-Robertson S
酵母中 AP 位点的旁路除了需要 Pol δ 跨损伤合成 DNA 聚合酶外，还需要 Rev1 蛋白。尽管 Rev1 最初在生物化学上被表征为 AP 位点旁路期间的 dCMP 转移酶，但这种活性在体内的相关性尚不清楚。目前的研究使用高度灵敏的移码和无义回复分析来监测从染色体 DNA 中切除尿嘧啶时产生的 AP 位点旁路。在移码回复测定中，未选择的碱基取代经常伴随着选定的突变，从而可以推断四个 dNMP 中的每一个与内源创建的 AP 位点相对的相对掺入情况。该测定的结果表明 dCMP 是插入尿嘧啶衍生 AP 位点对面的最常见的 dNMP，并证明 dCMP 插入绝对需要 Rev1 的催化活性。在互补的无义回复测定中，dCMP 插入同样取决于 Rev1 的 dCMP 转移酶活性。由于与尿嘧啶衍生的 AP 位点相对的 dAMP 插入不会恢复无义等位基因，因此无法检测到，因此在 Rev1 催化活性丧失时也可以检测到低水平的 dGMP 或 dTMP 插入。这些结果表明 Rev1 的催化活性具有生物学相关性，并且是绕过内源 AP 位点期间 dCMP 插入所必需的。
The bypass of AP sites in yeast requires the Rev1 protein in addition to the Pol ζ translesion synthesis DNA polymerase. Although Rev1 was originally characterized biochemically as a dCMP transferase during AP-site bypass, the relevance of this activity in vivo is unclear. The current study uses highly sensitive frameshift- and nonsense-reversion assays to monitor the bypass of AP sites created when uracil is excised from chromosomal DNA. In the frameshift-reversion assay, an unselected base substitution frequently accompanies the selected mutation, allowing the relative incorporation of each of the four dNMPs opposite endogenously created AP sites to be inferred. Results with this assay suggest that dCMP is the most frequent dNMP inserted opposite uracil-derived AP sites and demonstrate that dCMP insertion absolutely requires the catalytic activity of Rev1. In the complementary nonsense-reversion assay, dCMP insertion likewise depended on the dCMP transferase activity of Rev1. Because dAMP insertion opposite uracil-derived AP sites does not revert the nonsense allele and hence could not be detected, it also was possible to detect low levels of dGMP or dTMP insertion upon loss of Rev1 catalytic activity. These results demonstrate that the catalytic activity of Rev1 is biologically relevant and is required specifically for dCMP insertion during the bypass of endogenous AP sites.
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