Zhao Bo, Xie Zhongwen, Shen Huiyun, Wang Zhigang
Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA.
Nucleic Acids Res. 2004 Jul 29;32(13):3984-94. doi: 10.1093/nar/gkh710. Print 2004.
Abasic (AP) sites are major DNA lesions and are highly mutagenic. AP site-induced mutagenesis largely depends on translesion synthesis. We have examined the role of DNA polymerase eta (Poleta) in translesion synthesis of AP sites by replicating a plasmid containing a site-specific AP site in yeast cells. In wild-type cells, AP site bypass resulted in preferred C insertion (62%) over A insertion (21%), as well as -1 deletion (3%), and complex event (14%) containing multiple mutations. In cells lacking Poleta (rad30), Rev1, Polzeta (rev3), and both Poleta and Polzeta, translesion synthesis was reduced to 30%, 30%, 15% and 3% of the wild-type level, respectively. C insertion opposite the AP site was reduced in rad30 mutant cells and was abolished in cells lacking Rev1 or Polzeta, but significant A insertion was still detected in these mutant cells. While purified yeast Polalpha effectively inserted an A opposite the AP site in vitro, purified yeast Poldelta was much less effective in A insertion opposite the lesion due to its 3'-->5' proofreading exonuclease activity. Purified yeast Poleta performed extension synthesis from the primer 3' A opposite the lesion. These results show that Poleta is involved in translesion synthesis of AP sites in yeast cells, and suggest that an important role of Poleta is to catalyze extension following A insertion opposite the lesion. Consistent with these conclusions, rad30 mutant cells were sensitive to methyl methanesulfonate (MMS), and rev1 rad30 or rev3 rad30 double mutant cells were synergistically more sensitive to MMS than the respective single mutant strains.
无碱基（AP）位点是主要的DNA损伤，具有高度致突变性。AP位点诱导的诱变很大程度上取决于跨损伤合成。我们通过在酵母细胞中复制含有位点特异性AP位点的质粒，研究了DNA聚合酶η（Poleta）在AP位点跨损伤合成中的作用。在野生型细胞中，AP位点的跨越导致优先插入C（62%）而非A（21%），以及-1缺失（3%）和包含多个突变的复杂事件（14%）。在缺乏Poleta（rad30）、Rev1、Polζ（rev3）以及同时缺乏Poleta和Polζ的细胞中，跨损伤合成分别降至野生型水平的30%、30%、15%和3%。rad30突变细胞中与AP位点相对的C插入减少，而在缺乏Rev1或Polζ的细胞中则完全消除，但在这些突变细胞中仍能检测到显著的A插入。虽然纯化的酵母Polα在体外能有效地在AP位点相对处插入A，但纯化的酵母Poldelta由于其3'→5'校对核酸外切酶活性，在损伤相对处插入A的效率要低得多。纯化的酵母Poleta从损伤相对处的引物3'A进行延伸合成。这些结果表明Poleta参与酵母细胞中AP位点的跨损伤合成，并表明Poleta的一个重要作用是催化在损伤相对处插入A后的延伸。与这些结论一致，rad30突变细胞对甲基磺酸甲酯（MMS）敏感，而rev1 rad30或rev3 rad30双突变细胞对MMS的协同敏感性比各自的单突变菌株更高。
