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.

聚（ADP-核糖）聚合酶（PARP）通过与DNA结合而被激活 DNA链断裂并参与各种涉及DNA链的核过程 休息.这一修正提案旨在描述PARP在以下方面的确切作用： DNA复制修复和细胞凋亡。它基于一个总体假设， 带负电荷的长链 聚（ADP-核糖）（PAR）使靶DNA结合蛋白“DNA-疏水”， PAR的去除将恢复这些蛋白质的亲和力， DNA上的结合位点 特异性目的1A侧重于PARP作为多蛋白的组分的作用， 复制复合体（MRC），或“合成体”，由约40种蛋白质组成 并且能够忠实地复制SV 40的DNA MRCs来源于几个 癌细胞容易出错。几个MRC组件经过 聚（ADP-核糖基）化[p（ADP-R）n];使用来自PARP敲除小鼠的细胞 将用于确定p（ADP-R）n在MRC蛋白中的功能， DNA复制。为此，申请人最初将专注于一个专业 方面--从PARP敲除细胞纯化的MRC与对照相比， 表现出类似于癌细胞的缺陷，即容易出错的复制。 特异性目的1B集中于E2 F-1，其通常在细胞的S期诱导。 细胞周期，但在来自PARP敲除小鼠的细胞中不如此表达。的 PARP调节该基因启动子活性的机制将 因此要调查。此外，申请人将审查直接 PARP与E2 F-1启动子的直接相互作用，或与 与E2 F-1作用的其他信号蛋白，以及直接或间接 p（ADP-R）n对E2 F-1表达的影响。 具体目标2集中在早期和瞬时p（ADP-R）n的作用， 核蛋白，在上一个赠款期间描述，发生在早期 在细胞凋亡过程中，并且是许多后续变化所必需的 程序性细胞死亡的特征。的时空关系 这种瞬时p（ADP-R）n对染色质结构的变化和 核矩阵将被系统地检查和比较，部分，通过使用 PARP+/+细胞与PARP-/-细胞比较，以检测p（ADP-R）n在此水平的功能。 凋亡早期临界点。 Specific Aim 3的研究重点是转录因子和肿瘤抑制因子 p53是p（ADP-R）n在细胞凋亡过程中的主要靶点之一。申请人 在PAR早期爆发期间，p53被鉴定为p（ADP-R）n， 凋亡随后PAR从p53上裂解的时间与 其靶基因BAX的激活。这两者之间的机械关系 p53的可逆修饰及其反式激活活性和功能 将在体外和在整个细胞的情况下通过使用 凝胶迁移分析和其它p53应答基因的活性。