ENZYME MECHANISM OF POLY(ADP-RIBOSE) POLYMERASE

核腺苷二磷酸核糖聚合物与蛋白质结合对染色质的调节 高等真核生物的结构和功能。这些聚合物是 染色质结合的聚腺苷二磷酸核糖聚合酶(PARP)的合成 酶[EC 2.5.230]，利用BNAD作为ADP-核糖底物和 是由DNA链断裂激活的。这种由1014个氨基酸组成的蛋白质 SIZE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、延长和分支 均二聚(自改性)合成聚合物的反应 和与蛋白质受体的异二聚化(异构化)。 蛋白质-多聚(ADP-核糖基)似乎需要准确的 DNA复制过程中发生的DNA缺口和断裂的重新连接， 基因表达和DNA切除修复。基因与全长基因 PARP序列最近已可用。氨基酸序列 PARP的结构揭示了一种三方结构域结构。这些工具的功能 结构域是结合DNA(氨基末端)、自化位点(中心 区域)和底物结合(羧基末端)。然而， ADP-核糖聚合物中各结构域的分子协同作用 PARP的合成和酶机制尚待确定。 这项建议的具体目的是：1)剖析 聚(ADP-核糖)形成单独的引发，延伸， 和自化反应中的支化；2)表征 单(ADP-核糖)-组蛋白多肽的伸长和分支步骤 结构域作为受体(S)(异源聚(腺苷二磷酸核糖)化)；3) 测定ADP-的动力学机制和酸碱化学 PARP催化的核糖伸长反应；4)利用 利用PARP基因的定向突变体(SDM‘s)确定其催化作用 该酶的特定氨基酸基团。这项研究项目将 也识别和刻画(S)调节(S)的机制 它的蛋白质-蛋白质相互作用的多重酶活性 酶自身(分子间二聚体自化)以及 其他染色质成分(异源二聚体)。在具体目标1中， 将为每种反应开发具体的分析方法。入会仪式 反应将以纳摩尔浓度的NAD在 胍丁胺-(ADP-核糖)对链的特异性抑制作用 伸长率。伸长反应将用特定的 与ADP单体共价结合的自体修饰多肽片段 核糖(模拟引发胍丁胺-ADP-核糖)。此抑制器会阻止 ADP-核糖延伸受体的酶结合部位 PARP催化分支。类似的实验将在 含有受体的单(ADP-核糖基)多肽的存在 组蛋白H1和/或H_2B的结构域在特定目标中的作用#2.抑制研究 与苯甲酰胺(S)、胍丁胺-(腺苷二磷酸-核糖)和2‘dNAD也将携带 在具体目标#4中，PARP的SDM将 用来确认氨基酸残基的催化作用 参与酸碱化学合成ADP-核糖聚合物。

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.