ENZYME MECHANISM OF POLY(ADP-RIBOSE) POLYMERASE

核ADP-核糖聚合物结合蛋白质调节染色质 高等真核生物的结构和功能。 这些聚合物 由聚（ADP-核糖）聚合酶（PARP）合成， 酶[EC 2.5.230]，其利用BNAD作为ADP-核糖底物， 被DNA链断裂激活 这种蛋白质由1014个氨基酸组成， 大小利用DNA结合蛋白作为（ADP-核糖）n受体。 它还 催化ADP-核糖链的起始、延伸和分支 通过均二聚（自改性）的聚合物合成反应 和与蛋白质受体的异源二聚化（异源修饰）。 蛋白质-聚（ADP-核糖基）化似乎是准确的 在DNA复制过程中发生的DNA上的切口和断裂的重新连接， 基因表达和DNA切除修复。 该基因和全长cDNA PARP序列最近已经可用。 氨基酸序列 的PARP揭示了一个三方域结构。 这些功能 结构域是结合DNA（氨基末端），自动修饰位点（中央 区域）和底物结合（羧基末端）。 但 ADP-核糖聚合物中各结构域的分子协同作用 PARP的合成和酶机制仍有待建立。 这一建议的具体目标是：1）剖析 聚（ADP-核糖）转化为起始，延伸， 和支化; 2）表征 单（ADP-核糖）-组蛋白肽的延伸和分支步骤 结构域作为受体（异源聚（ADP-核糖基）化）; 3） 确定ADP的动力学机制和酸基化学， PARP催化的核糖延伸反应;和4）利用位点- PARP基因的定向突变体（SDM），以确定其催化作用 酶的特定氨基酸组的。 该研究项目将 还确定和表征调节 这种蛋白质-蛋白质相互作用的多种酶活性 酶与自身（分子间二聚体自修饰）以及 其他染色质成分（异二聚化）。 在具体目标1中， 将开发用于每种反应的特定分析。 启动 反应将在NAD的纳摩尔浓度下表征， 胍丁胺-（ADP-核糖）的存在，以特异性抑制链 伸长率 延伸反应将在特定的条件下进行。 与单体ADP共价结合的自修饰肽片段 核糖（模拟起始胍丁胺-ADP-核糖）。 这种抑制剂阻断了 ADP-核糖延伸受体的酶结合位点， PARP催化分支。 类似的实验将在 存在含有受体的单（ADP-核糖基）化肽 组蛋白H1和/或H2 b的结构域在特定目标#2中。 抑制研究 与苯甲酰胺，胍丁胺-（ADP-核糖）和2 'dNAD也将携带 #36825;的具体目标。 在具体目标#4中，PARP的SDM将 用于确认氨基酸残基的催化作用 参与ADP-核糖聚合物合成的酸基化学。

Nuclear ADP-ribose polymers bound to proteins modulate chromatin structure and function in higher eucaryotes. These polymers are synthesized by poly (ADP-ribose) polymerase (PARP), a chromatin-bound enzyme [EC 2.5.230], that utilizes BNAD as the ADP-ribose substrate and is activated by DNA-strand breaks. This protein of 1014 amino acids in size utilizes DNA-binding proteins as (ADP-ribose)n acceptors. It also catalyzes the ADP-ribose chain initiation, elongation, and branching reactions of polymer synthesis via homodimerization (automodification) and heterodimerization with protein acceptors (heteromodification). Protein-poly(ADP-ribosyl)ation appears to be required for the accurate rejoining of nicks and breaks on DNA that occur during DNA-replication, gene expression, and DNA-excision repair. The gene and full cDNA sequence of PARP have recently become available. The amino acid sequence of PARP reveals a tripartite domain structure. The function of these domains is to bind DNA (amino-terminus), automodification sites (central region), and substrate binding (carboxy-terminus). However, the molecular cooperation of the various domains in ADP-ribose polymer synthesis and the enzyme mechanisms of PARP remain to be established. The specific aims of this proposal are to: 1) dissect the synthesis of poly (ADP-ribose) into individual reactions of initiation, elongation, and branching in the automodification reaction; 2) characterize the elongation, and branching steps with mono(ADP-ribose)-histone peptide domains as the acceptor(s) (heterologous poly(ADP-ribosyl)ation); 3) determine the kinetic mechanics and the acid-based chemistry of the ADP- ribose elongation reaction catalyzed by PARP; and 4) utilize site- directed mutants (SDM's) of the PARP gene to determine the catalytic role of specific amino acid groups of the enzyme. This research project will also identify and characterize the mechanism(s) that modulate(s) the multiple enzymatic activities of protein-protein interactions of this enzyme with itself (intermolecular dimeric automodification) as well as other chromatin components (heterodimerization). In specific aim #1, specific assays for each reaction will be developed. The initiation reaction will be characterized at nanomolar concentrations of NAD in the presence of agmatine-(ADP-ribose) to specifically inhibit chain elongation. The elongation reaction will be performed with specific automodification peptide fragments covalently bound to monomeric ADP- ribose(mock initiation agmatine-ADP-ribose). This inhibitor blocks the enzyme binding site for the ADP-ribose elongation acceptor which forces PARP to catalyze branching. Similar experiments will be performed in the presence of mono(ADP-ribosyl)ated-peptides containing the acceptor domains of histone H1 and/or H2b in specific aims #2. Inhibition studies with benzamide(s), Agmatine-(ADP-ribose) and 2'dNAD will also be carried out to achieve specific aim #3. In specific aim#4, SDM's of PARP will be utilized to confirm the catalytic role of the amino acid residues involved in the acid-based chemistry of ADP-ribose polymer synthesis.