HUMAN HOMOLOGS OF YEAST REV GENES--ROLE IN MUTAGENESIS

这项拟议中的研究涉及一个非常基本的重要问题：“人类细胞中诱导的大多数突变是否是由于特殊蛋白质进行易错的跨损伤合成以绕过阻碍分叉进展的DNA损伤而产生的？ 直到现在，这个问题都没有答案。突变被认为对人类细胞的肿瘤转化有显著贡献。 如果自发产生的或外源性DNA损伤在S期复制之前没有修复，并且未修复的损伤干扰叉进展，则认为调用了称为跨损伤旁路的过程。 在细菌和低等真核生物中，这种跨损伤合成依赖于一组蛋白质，这些蛋白质至少部分不同于用于正常染色体复制的蛋白质。 然而，在哺乳动物细胞中的过程的性质是未知的。 最近，C.W.劳伦斯和他在罗切斯特大学的研究小组克隆了S.酿酒酵母REV 3和Rev 1基因。 使用我们的无限寿命的衍生物，近二倍体，核型稳定的细胞株，MSU-1.2，表达反义hsREV 3的四环素启动子的控制下，我们进行了一项试点研究比较的频率突变诱导的UV和MSU-1.2细胞，不表达这种反义。 在具有反义hsREV的细胞中诱导的突变体的频率显著降低。 这些结果虽然是初步的，但提出了这样的可能性，即由各种试剂在人类细胞中诱导的大多数突变是由scRev 3 p + scRev 7 p（聚合酶zeta）的人类等同物和scREV 1基因产物的跨损伤合成引起的。 我们将在表达或不表达反义hsREV 3和hsREV 1的人细胞株组以及内源性基因已或未被突变消除的细胞中检验这一假设。 待测试的试剂包括UV、BPDE、1-NOP、NAcO-AAF、MNU和ENU以及Co 60。 将比较这些细胞株在HPRT基因中诱导突变的频率和谱。 还将比较来自这些相同菌株的无细胞提取物绕过RF I DNA中的损伤的能力以及LacZ-α基因中诱导的突变的频率和谱，所述相同菌株能够复制含有由前四种试剂诱导的损伤的M13 mp2噬菌体RF I。

The proposed research deals with a very fundamental important question: "Do the majority of the mutations induced in human cells arise as the result of special proteins carrying out error- prone translesion synthesis to bypass DNA damage that blocks fork progression? Until now, this question could not be answered. Mutations are considered to contribute significantly to the neoplastic transformation of human cells. If spontaneously arising or exogenous DNA damage is not repaired before S-phase replication, and the unrepaired damage interferes with fork progression, a process known as translesion bypass is considered to be invoked. In bacteria and lower eukaryotes, such translesion synthesis depends upon a set of proteins that differ, at least in part, from those used for normal chromosome replication. However, the nature of the process in mammalian cells is unknown. Very recently, C.W. Lawrence and his group at the Univ. of Rochester cloned the human homolog of the S. cerevisiae REV3 and Rev1 genes. Using a derivative of our infinite life span, near-diploid, karyotypically-stable cell strain, MSU-1.2, that expresses antisense hsREV3 under the control of a tetracycline promoter, we carried out a pilot study comparing the frequency of mutations induced by UV and in MSU-1.2 cells that do not express this antisense. The frequency of mutants induced in the cells with antisense hsREV was significantly lower. These results, although preliminary, raise the possibility that the majority of mutations induced in human cells by a variety of agents result from translesion synthesis by the human equivalent of scRev3p + scRev7p (polymerase zeta) and the scREV1 gene product. We will test this hypothesis in sets of human cell strains that express or do not express antisense hsREV3 and hsREV1 and cells in which the endogenous genes have or have not been eliminated by mutations. The agents to be tested include UV, BPDE, 1-NOP, N AcO-AAF, MNU and ENU, and Co60. These cell strains will be compared for the frequency and spectra of mutations induced in the HPRT gene. Cell-free extracts from these same strains, capable of replicating M13mp2 phage RF I containing damage induced by the first four agents will also be compared for the ability to by-pass the damage in the RF I DNA and for the frequency and spectra of mutations induced in the LacZ-alpha gene.