Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 01239, USA.
Genetics. 2011 Jan;187(1):21-35. doi: 10.1534/genetics.110.124172. Epub 2010 Oct 26.
A cell's ability to tolerate DNA damage is directly connected to the human development of diseases and cancer. To better understand the processes underlying mutagenesis, we studied the cell's reliance on the potentially error-prone translesion synthesis (TLS), and an error-free, template-switching pathway in Saccharomyces cerevisiae. The primary proteins mediating S. cerevisiae TLS are three DNA polymerases (Pols): Rev1, Pol ζ (Rev3/7), and Pol η (Rad30), all with human homologs. Rev1's noncatalytic role in recruiting other DNA polymerases is known to be important for TLS. However, the biological significance of Rev1's unusual conserved DNA polymerase activity, which inserts dC, is much less well understood. Here, we demonstrate that inactivating Rev1's DNA polymerase function sensitizes cells to both chronic and acute exposure to 4-nitroquinoline-1-oxide (4-NQO) but not to UV or cisplatin. Full Rev1-dependent resistance to 4-NQO, however, also requires the additional Rev1 functions. When error-free tolerance is disrupted through deletion of MMS2, Rev1's catalytic activity is more vital for 4-NQO resistance, possibly explaining why the biological significance of Rev1's catalytic activity has been elusive. In the presence or absence of Mms2-dependent error-free tolerance, the catalytic dead strain of Rev1 exhibits a lower 4-NQO-induced mutation frequency than wild type. Furthermore, Pol ζ, but not Pol η, also contributes to 4-NQO resistance. These results show that Rev1's catalytic activity is important in vivo when the cell has to cope with specific DNA lesions, such as N(2)-dG.
细胞耐受 DNA 损伤的能力与人类疾病和癌症的发展直接相关。为了更好地理解诱变的潜在过程，我们研究了细胞对潜在易错的跨损伤合成（TLS）和酿酒酵母中无差错、模板切换途径的依赖。介导酿酒酵母 TLS 的主要蛋白质是三种 DNA 聚合酶（Pols）：Rev1、Pol ζ（Rev3/7）和 Pol η（Rad30），它们都有人类同源物。Rev1 在招募其他 DNA 聚合酶方面的非催化作用对 TLS 很重要。然而，Rev1 异常保守的 DNA 聚合酶活性插入 dC 的生物学意义要小得多。在这里，我们证明了失活 Rev1 的 DNA 聚合酶功能会使细胞对慢性和急性暴露于 4-硝基喹啉-1-氧化物（4-NQO）敏感，但对 UV 或顺铂不敏感。然而，完全依赖 Rev1 的对 4-NQO 的抗性也需要额外的 Rev1 功能。当通过缺失 MMS2 破坏无差错耐受时，Rev1 的催化活性对 4-NQO 抗性更为重要，这可能解释了为什么 Rev1 的催化活性的生物学意义一直难以捉摸。在存在或不存在 Mms2 依赖的无差错耐受的情况下，催化失活的 Rev1 菌株在 4-NQO 诱导下的突变频率低于野生型。此外，Pol ζ，而不是 Pol η，也有助于 4-NQO 抗性。这些结果表明，当细胞必须应对特定的 DNA 损伤，如 N(2)-dG 时，Rev1 的催化活性在体内是重要的。
