CARCINOGENS AND CHROMATIN STRUCTURE AND FUNCTION

聚(ADP-核糖)聚合酶(PARP)通过与DNA结合而被激活 链断裂并参与涉及DNA链的各种核过程 休息一下。这项修改后的提案旨在描述PARP在 DNA复制-修复和细胞凋亡。它是基于总体假设的 长的带负电荷的链的共价连接 聚(ADP-核糖)(PAR)使靶DNA结合蛋白“DNA恐惧症”和 PAR的去除将恢复这些蛋白质对 DNA上的结合位点。 具体目标1侧重于PARP作为多蛋白的一个组成部分的作用 复制复合体(MRC)，或“合成体”，由大约40种蛋白质组成 并且能够忠实地复制SV40 DNA。MRCS派生自若干 癌细胞很容易出错。几个MRC组件经历了 聚(ADP-核糖基)聚合[p(ADP-R)n]；PARP基因敲除小鼠细胞的应用 将被用来确定p(腺苷二磷酸核苷-受体)n对mrc蛋白的功能(S) DNA复制。为此，申请者最初将专注于一个专业 方面--无论是从PARP基因敲除细胞和对照细胞中纯化的MRC， 表现出类似于癌细胞的缺陷，即容易错误复制。 具体目标1B集中在E2F-1，通常是在S阶段诱导的 细胞周期，但在PARP基因敲除小鼠的细胞中不表达。这个 PARP调节该基因启动子活性的机制 因此被调查。此外，申请者还将直接审查 PARP与E2F-1启动子的直接相互作用或联合作用 与E2F-1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 具体目标2集中于早期和瞬时的p(ADP-R)n的作用 核蛋白，在最后一次授权期内描述，发生在早期 在细胞凋亡过程中，这是许多后续变化所必需的 以细胞程序性死亡为特征。时间和空间上的关系 这种瞬时的p(ADP-R)n对染色质结构的变化和 将系统地检查和比较核矩阵，部分方法是使用 PARP+/+细胞与PARP-/-细胞相比，检测p(ADP-R)n的功能 细胞凋亡的早期临界点。 特异性靶点3的焦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的早期爆发期间，短暂地确定了P53是p(ADP-R)n 细胞凋亡。随后从P53上切割PAR在时间上与 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 将在体外和整个细胞的环境中通过使用 凝胶漂移试验和其他P53反应基因的活性。

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.