CARCINOGENS AND CHROMATIN STRUCTURE AND FUNCTION

致癌物和染色质结构与功能

• 批准号: 6375614
• 负责人: MARK E SMULSON
• 金额: $ 24.2万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-06-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375614
• 关键词: ADP ribosylation; DNA directed DNA polymerase; DNA repair; DNA replication; adenosine diphosphate; apoptosis; carcinogen testing; chemical carcinogen; chemical carcinogenesis; chemical structure function; chromatin; enzyme activity; genetic recombination; genetically modified animals; laboratory mouse; mutagen testing; nucleic acid structure; p53 gene /protein; pentosyltransferase; pulsed field gel electrophoresis; transcription factor

Poly(ADP-ribose) polymerase (PARP) is activated by binding to DNA strand breaks and contributes to various nuclear processes involving DNA strand breaks. This amended proposal aims to characterize the precise roles of PARP in DNA replication-repair and in apoptosis. It is based on the overall hypothesis that the covalent attachment of long, negatively charged chains of poly(ADP-ribose) (PAR) renders target DNA-binding proteins "DNA-phobic" and that removal of PAR would then restore the affinity of these proteins for binding sites on DNA. Specific Aim 1A focuses on the role of PARP as a component of the multiprotein replication complex (MRC), or "synthesome", which comprises about 40 proteins and is able to replicate, faithfully, SV40 DNA. MRCs derived from several cancer cell are error-prone. Several MRC components undergo poly(ADP-ribosyl)ation [p(ADP-R)n]; the use of cells from PARP knockout mice will be used to determine the function(s) of p(ADP-R)n on MRC proteins during DNA replication. To this end, the applicant will initially focus on one major facet--whether the MRC, purified from PARP knockout cells versus controls, exhibits a defect similar to cancer cells, that of error-prone replication. Specific Aim 1B focuses on E2F-1, which is normally induced during S phase of the cell cycle, but is not so expressed in cells from PARP knockout mice. The mechanism by which PARP regulates the promoter activity of this gene will therefore be investigated. In addition, the applicant will examine the direct interaction of PARP with the E2F-1 promoter directly, or in combination with other signal proteins that act with E2F-1, as well as direct or indirect effects of p(ADP-R)n on expression of E2F-1. Specific Aim 2 is focused on the role of the early and transient p(ADP-R)n of nuclear proteins, described during the last grant period, which occurs early during apoptosis and is required for many of the subsequent changes characteristic of programmed cell death. The temporal and spatial relations of this transient p(ADP-R)n to changes in the structure of chromatin and the nuclear matrix will be examined systematically and compared, in part, by using PARP+/+ cells vs. PARP-/- cells to detect the function of p(ADP-R)n at this early critical point of apoptosis. The focus of Specific Aim 3 is the transcription factor and tumor suppressor p53, one of the major targets of p(ADP-R)n during apoptosis. The applicant identified p53 to be p(ADP-R)n briefly during the early burst of PAR in apoptosis. The subsequent cleavage of PAR from p53 coincides temporally with the activation of its target gene BAX. The mechanistic relation between this reversible modification of p53 and its transactivation activity and function will be determined in vitro and in the context of the whole cell by the use of gel shift assays and the activity of other p53 responsive genes.

通过与DNA结合激活聚（ADP-核糖）聚合酶（PARP） 链断裂并有助于涉及DNA链的各种核过程 休息。这项修订的建议旨在表征PARP的精确作用 DNA复制修复和凋亡。它基于整体假设 长长，负电荷的链的共价附件 聚（ADP-核糖）（PAR）渲染目标DNA结合蛋白“ DNA-恐惧症”和 然后去除PAR将恢复这些蛋白质的亲和力 DNA上的结合位点。 特定的目标1A专注于PARP作为多蛋白的组成部分的作用 复制复合物（MRC）或“合成”，该复制复合物包括约40种蛋白 并能够忠实地复制SV40 DNA。 MRC来自几个 癌细胞容易出错。几个MRC组件发生 聚（ADP-核糖基）[P（ADP-R）N];使用PARP基因敲除小鼠的细胞 将用于确定在MRC蛋白上的P（ADP-R）N的功能 DNA复制。为此，申请人最初将专注于一个主要 方面 - MRC是否从PARP基因敲除单元与对照组中纯化， 表现出类似于癌细胞的缺陷，即易于错误的复制。 特定的目标1B专注于E2F-1，通常在S期诱导的E2F-1 细胞周期，但在PARP基因敲除小鼠的细胞中不太表达。这 PARP调节该基因启动子活性的机制将 因此被调查。此外，申请人将检查直接 PARP与E2F-1启动子的相互作用，或与E2F-1启动子的相互作用，或与 与E2F-1一起起作用的其他信号蛋白以及直接或间接的 P（ADP-R）N对E2F-1表达的影响。 特定目标2的重点是早期和瞬态P（ADP-R）N的作用 核蛋白，在上一个赠款期间描述，早期发生 在凋亡期间，随后的许多变化是必需的 程序性细胞死亡的特征。的时间和空间关系 该瞬态P（ADP-R）N与染色质结构的变化和 将系统地检查核矩阵，并通过使用 PARP+/+细胞与PARP - / - 细胞在此检测P（ADP-R）N的功能 凋亡的早期关键点。 特定目标3的重点是转录因子和肿瘤抑制剂 p53，凋亡过程中P（ADP-R）N的主要靶标之一。申请人 在PAR早期爆发期间，将p53识别为p（adp-r）n 凋亡。随后从p53的PAR裂解与 其靶基因BAX的激活。这之间的机械关系 p53的可逆修改及其反式激活活性和功能 将通过使用 凝胶转移测定和其他p53反应性基因的活性。