HUMAN HOMOLOGS OF YEAST REV GENES--ROLE IN MUTAGENESIS

酵母 Rev 基因的人类同源物——在诱变中的作用

• 批准号: 6518145
• 负责人: VERONICA M MAHER
• 金额: $ 23.59万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6518145
• 关键词: DNA damage; DNA repair; Saccharomyces cerevisiae; adduct; alkylating agents; antisense nucleic acid; cell free system; fibroblasts; fungal genetics; gamma radiation; gene mutation; genetic strain; human genetic material tag; human tissue; mutagens; photochemistry; point mutation; radiation genetics; tissue /cell culture; ultraviolet radiation

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.

拟议的研究涉及一个非常基本的重要问题：“由于特殊的蛋白质执行误差 - 容易发生的transe综合绕过DNA损伤，会导致人类细胞中引起的大多数突变会产生，从而阻止了叉子的进展？直到现在，直到现在，该问题才能在人类造成的肿瘤变化或造成较大的造成的造成肿瘤的造成的造成或散发性的造成的疾病。 S期复制和未修复的损害干扰了叉子的进展，被称为Translesion旁路的过程被认为是在细菌中调用的。他在罗切斯特大学的小组克隆了酿酒酵母Rev3和Rev1基因的人类同源物。 使用我们无限寿命的衍生物，近二倍体，核型稳定的细胞菌株MSU-1.2（在四环素启动子的控制下表达反义HSREV3），我们进行了一项试验研究，比较了UV和MSU-1.2细胞中未表达这种反态的突变频率，该研究比较了突变的频率。 反义HSREV在细胞中诱导的突变体的频率显着降低。 这些结果虽然初步，但却增加了人类细胞中大多数突变的可能性，这些突变是由screv3p + screv7p（聚合酶zeta）和screv1基因产物的人类等效物进行跨质量合成而引起的。 我们将在表达或不表达反义HSREV3和HSREV1的一组人细胞菌株中检验该假设，以及内源基因所具有或未通过突变消除的细胞。 要测试的代理包括UV，BPDE，1-NOP，N ACO-AAF，MNU和ENU以及CO60。 这些细胞菌株将与HPRT基因中诱导的突变的频率和光谱进行比较。 还将比较这些相同菌株中的无细胞提取物，能够复制M13MP2噬菌体RF I含有前四个药物诱导的损伤的M13MP2噬菌体i，也将比较绕开RF I DNA中损伤的能力以及LACZ-Alpha基因中引起的突变的频率和光谱。