HUMAN HOMOLOGS OF YEAST REV GENES--ROLE IN MUTAGENESIS

拟议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由于特殊蛋白质进行容易出错的翻译合成，以绕过阻止叉子进展的DNA损伤而产生的吗？”直到现在，这个问题还没有答案。突变被认为对人类细胞的肿瘤转化有重要贡献。如果自发产生的或外源性DNA损伤在s期复制之前没有得到修复，并且未修复的损伤干扰了叉的进展，则被认为调用了一个称为翻译旁路的过程。在细菌和低等真核生物中，这种转译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，哺乳动物细胞中这一过程的性质尚不清楚。最近，罗切斯特大学的C.W.劳伦斯和他的团队克隆了酿酒葡萄球菌REV3和Rev1基因的人类同源物。利用我们的无限寿命，近二倍体，核型稳定的细胞株MSU-1.2的衍生物，在四环素启动子的控制下表达反义hsREV3，我们进行了一项初步研究，比较紫外线诱导的突变频率和不表达反义hsREV3的MSU-1.2细胞。反义hsREV诱导的突变体频率明显降低。这些结果虽然是初步的，但提出了一种可能性，即各种药物在人类细胞中诱导的大多数突变是由人类等效的scRev3p + scRev7p（聚合酶zeta）和scREV1基因产物的翻译合成引起的。我们将在表达或不表达反义hsREV3和hsREV1的人类细胞株以及内源基因已或未被突变消除的细胞中验证这一假设。待测试剂包括UV、BPDE、1-NOP、N AcO-AAF、MNU和ENU、Co60。这些细胞株将比较在HPRT基因诱导突变的频率和频谱。来自这些菌株的无细胞提取物，能够复制含有前四种药物诱导的损伤的M13mp2噬菌体RF I，也将比较绕过RF I DNA损伤的能力以及lacz - α基因诱导的突变的频率和谱。

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.