Mechanisms of Inducible Error Prone DNA Replication

诱导错误 DNA 复制机制

• 批准号: 6633218
• 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/6633218
• 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(申请人提供)：诱变剂通过两个不同的 途径：第一个途径是通过DNA损伤的直接错误复制 由诱变剂造成的。第二种是间接途径，在这种途径中， 诱变剂改变细胞生理，例如不仅增强诱变 在触发(同源)DNA损伤处，也在其他(异源)DNA处 损伤存在于基因组和未受损的细胞中。对该等情况的调查 基础水平上的“瞬时突变”表型是必要的 理解遗传变异的潜在机制 环境条件，并对医疗保健问题产生影响，如 癌细胞中多种突变的矛盾积累，尽管正常情况下 突变率低(10-6个/基因/代)，抗药性发展迅速 敬抗癌药物。UVM(用于紫外线诱变的调制)是最近出现的一种 描述了RecA非依赖的DNA损伤诱导的突变反应 在我们实验室发现的大肠杆菌线圈。UVM促进突变 2类非指导性突变DNA损伤，如3，N4-乙胞嘧啶(EC) 以与SOS无关的方式，在1类非指导性突变病变中 例如以依赖于SOS的方式的AP站点。其中的工作假说 有人提出，大肠杆菌中的DNA损伤会引发两种平行的现象，即， SOS和UVM，在可诱导的损伤中发挥互补的损伤依赖作用 诱变。UVM途径的遗传和生化基础将是 这项提案通过两个具体目标来追求。目标1：我们将确定和 通过以下方式描述UVM途径中涉及的基因 接近了。(A)根据以下条件对UVM缺陷突变体进行两步筛选 对DNA损伤剂的敏感性，以及对UVM的影响。(B)确定大多数基因 它的转录在使用Affymetrix诱导UVM时发生改变 基因芯片技术。目标2：我们将检验UVM是由以下因素介导的假设 一种瞬间改变的DNA聚合酶III(POL-ILI) 接近了。(A)测定体内和体外诱导IJVM的动力学。 (B)分析UVM诱导的dna E(Ts)突变体(缺陷)中容易出错的复制 对于POL-I11)在允许和限制的条件下。(C)净化和 描述UVM诱导的细胞中容易出错的聚合酶活性。