ISOLATION AND CHARACTERIZATION OF HUMAN GENES AFFECTING CHROMOSOME METABOLISM

工作概述：功能基因组学研究人类基因编码蛋白的结构和功能。我们在酵母中开发了基于遗传学的方法来了解可能在DNA和染色体代谢以及基因组稳定性中起作用的人类基因的功能。人类细胞周期蛋白A1和A2基因改变了酵母复制突变体的表型。我们还确定了表达hRad51p导致合成致死的遗传背景，提供了hRad51p的分子特征及其与克隆到酵母中的其他人类蛋白的相互作用。BRCA1和p53)。参与DNA复制和修复的人Fen1皮瓣内切酶能够补充酵母rad27突变体的突变原敏感性，遗传不稳定性以及与修复和复制突变体的合成致命相互作用。我们发现了一种生物化学无活性形式的Fen1，具有显性的遗传毒性作用。当人和酵母PCNA结合域交换时，Fen1在酵母中的遗传影响更强。我们的研究定义了一个系统来研究人类FEN1中潜在的有害突变。在与Kunkel实验室的合作中，基于酵母中共同过表达的人类蛋白的强突变效应，开发了用于人类Msh2/Msh6蛋白功能分析的酵母模型。人类p53的功能分析一直在使用已建立的和新开发的酵母测定法进行。从肿瘤样本和细胞系中直接克隆酵母的p53基因的转激活潜力已经被评估。我们利用酵母中人类p53的可变表达和DNA结合域和四聚域的随机PCR诱变来筛选具有新表型的p53等位基因。发现P53突变体在适度表达水平上杀死酵母。其中一种p53毒素突变体阻止了人类细胞的生长。第二次筛选旨在分离与野生型相比具有更高的交易激活潜力的p53变体。这两种类型的p53突变等位基因将在旨在解剖复杂的p53下游通路和开发癌症治疗的研究中有用。-酵母菌，人类基因，表型破坏，DNA重组，复制，DNA修复

Summary of Work: Functional genomics elucidates structure and function of proteins encoded by human genes. We developed genetic-based approaches in yeast to understand functions of human genes that may have a role in DNA and chromosome metabolism and genome stability. The human cyclins A1 and A2 genes alter phenotype in a yeast replication mutant. We also determined a genetic background in which expressed hRad51p results in synthetic lethality, providing for molecular characterization of hRad51p and its interactions with other human proteins cloned into yeast (eg. BRCA1 and p53). The human Fen1 flap endonuclease involved in DNA replication & repair was able to complement a yeast rad27 mutant for mutagen sensitivity, genetic instability and synthetic lethal interactions with repair and replication mutants. We identified a biochemically inactive form of Fen1 with dominant genotoxic effects. The genetic impacts of Fen1 in yeast is stronger when the human and yeast PCNA binding domains are exchanged. Our study defines a system to study potentially deleterious mutations in human FEN1. In collaborations with the Kunkel lab, a yeast model for functional analysis of human Msh2/Msh6 protein was developed, based on the strong mutator effect of the co-overexpressed human proteins in yeast. Functional analysis of human p53 has been pursued using established and newly developed yeast assays. The transactivation potential of p53 genes cloned directly in yeast from tumor samples and cell lines has been assessed. We exploited variable expression of human p53 in yeast and random PCR mutagenesis of the DNA binding and tetramerization domains to develop screens for p53 alleles with novel phenotypes. p53 mutants were identified that kill yeast at modest expression levels. One of these p53 toxin mutants prevented growth of human cells. A second screen was aimed at isolating p53 variants with an increased transactivation potential compared to the wild type. Both categories of p53 mutant alleles will be useful in studies aimed at dissecting the complex p53 downstream pathways and in developing cancer therapies. - Yeast, Human Gene, Phenotype Disruption, DNA Recombination, Replication, DNA Repair