Mechanisms of Inducible Error Prone DNA Replication

诱导错误 DNA 复制机制

• 批准号: 6513051
• 负责人: M. ZAFRI HUMAYUN
• 金额: $ 27.07万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6513051
• 关键词: DNA damage; DNA directed DNA polymerase; DNA replication; Escherichia coli; affinity chromatography; bacterial genetics; bacterial proteins; carcinogen testing; chemical carcinogen; enzyme activity; gel filtration chromatography; gene induction /repression; methane sulfonate; mutagen testing; mutagens; radiation carcinogen; radiation genetics; transposon /insertion element; ultraviolet radiation

DESCRIPTION (provided by applicant): Mutagens act through two distinct pathways: the first is through the direct misreplication of DNA damage inflicted by the mutagen. The second is an indirect pathway in which the mutagens alter the cellular physiology such as to enhance mutagenesis not only at the triggering (cognate) DNA lesions, but also at other (heterologous) DNA damage present in the genome, and at undamaged cells. Investigation of such "transient mutator" phenotypes at a fundamental level is necessary for understanding the mechanisms underlying genetic variability in response to environmental conditions, and has impact on healthcare issues such as the paradoxical accrual of multiple mutations in cancerous cells despite normally low mutation rates (10-6/gene/generation), and rapid development of resistance to anti-cancer agents. UVM (for UV modulation of mutagenesis) is a recently described recA-independent DNA damage-inducible mutagenic response in Escherichia coil discovered in our laboratory. UVM promotes mutagenesis at Class 2 noninstructive mutagenic DNA lesions such as 3,N4-ethenocytosine (EC) in an SOS-independent manner, and at Class 1 noninstructive mutagenic lesions such as AP sites in an SOS-dependent manner. The working hypothesis in this proposal is that DNA damage in E. coli triggers two parallel phenomena, namely, SOS and UVM, that play complementary lesion-dependent roles in inducible mutagenesis. The genetic and biochemical basis of the UVM pathway will be pursued in this proposal through two specific aims. Aim 1: We will identify and characterize genes involved in the UVM pathway through the following approaches. (a) Apply a 2-step screen for UVM-defective mutants based on sensitivity to DNA damaging agents, and effect on UVM. (b) Identify most genes whose transcription is altered upon UVM induction by using the Affymetrix GeneChip technology. Aim 2: We will test the hypothesis that UVM is mediated by a transiently altered DNA polymerase III (pol-IlI) through following approaches. (a) Determine the kinetics of IJVM induction in vivo and in vitro. (b) Analyze error-prone replication in UVM-induced dnaE(Ts) mutants (defective for pol-Ill) under permissive and restrictive conditions. (c) Purify and characterize the error-prone polymerase activity in UVM-induced cells.

描述（由申请人提供）：诱变剂通过两种不同的 途径：第一种是通过DNA损伤的直接错误复制 是变异原造成的第二种是间接途径， 诱变剂改变了细胞生理学， 在触发（同源）DNA损伤，但也在其他（异源）DNA 基因组中存在的损伤，以及未受损的细胞。调查此类 在基础水平上的“瞬时增变因子”表型对于 了解遗传变异的潜在机制， 环境条件，并对医疗保健问题，如 癌细胞中多个突变的反常累积，尽管正常 突变率低（10-6/基因/代），抗性发展迅速 到抗癌剂。UVM（紫外诱变调制）是一种新的诱变方法， 描述了recA非依赖性DNA损伤诱导的诱变反应， 大肠杆菌在我们实验室发现。UVM促进诱变， 2类非指示性致突变DNA损伤，如3，N4-乙烯胞嘧啶（EC） 以SOS非依赖性方式，在1级非指示性致突变性病变中 例如以依赖于SOS的方式的AP站点。这方面的工作假设是 推测E.大肠杆菌引发了两种平行现象，即， SOS和UVM，在诱导型中发挥互补的病变依赖性作用， 诱变UVM途径的遗传和生化基础将是 在这一建议中，有两个具体目标。目标1：我们将确定和 通过以下方式表征参与UVM途径的基因 接近。(a)应用基于以下的两步筛选UV缺陷突变体： 对DNA损伤剂的敏感性和对UVM的影响。(b)识别大多数基因 其转录在UVM诱导后通过使用Affyphase 基因芯片技术目的2：我们将检验UVM由以下因素介导的假设： 瞬时改变的DNA聚合酶III（pol-III），通过以下步骤： 接近。(a)确定体内和体外IJVM诱导的动力学。 (b)分析UV诱导的dnaE（Ts）突变体（缺陷型）中的易错复制 对于pol-III）在许可和限制条件下。(c)净化和 表征在UVM诱导的细胞中的易错聚合酶活性。