Factors Influencing Genetic Transcription Initiation And Termination

RNA形成部分的研究旨在了解和利用细胞DNA复制所涉及的过程，艾滋病毒复制与这些细胞事件的关系，以及如何将这些信息用于治疗目的。我们正在研究DNA复制和/或转录过程中发生的RNA/DNA杂交体的形成和分解。核糖核酸酶H是参与RNA/DNA杂交物RNA去除的重要酶，与细胞内DNA复制和HIV病毒基因组向DNA转化过程中的复制密切相关。细胞的RNase H和HIV共享共同的酶机制，即利用相似的蛋白质结构裂解RNA。改变特定疾病相关基因水平的药物正在开发中，以利用细胞内的RNase H。调节RNase H的表达可增强药物的疗效。分子遗传学、生物化学和小鼠动物模型被用于这些努力。 在转录过程中，RNA/DNA杂交物可以形成并通常被RNaseH切割。RNaseH通过与RNA的多次接触以及与DNA链的相互作用，使用双金属离子机制识别并特异性切割杂交物的RNA。我们现在知道，真核RNase H比细菌酶更复杂，真核RNase H1的酶活性与RNA/DNA杂交物结合时形成一个复合体，使该酶在降解某些类型的RNA/DNA杂交物时更有效。在与NCI-Frederick的研究小组的合作下，我们已经检查了RNases H的抑制剂，这些抑制剂可能被证明在艾滋病毒-艾滋病治疗中有用。从我们早期的工作表明小鼠RNaseH1对发育至关重要，我们知道有专门针对HIV-AIDS RNaseH而不是细胞酶的药物是重要的。潜在药物的筛选现在包括使用在我们实验室分离的人RNaseH1。 小鼠RNaseH1从单个信使RNA翻译而来，产生相同的蛋白质驻留在细胞核和线粒体中。这两种形式的产生取决于我们发现在许多真核生物和其他生物中保守的翻译过程，以及针对细胞核和线粒体的DNA交易蛋白。 AGS是一种综合征，其特征是一种严重的神经系统疾病，模仿宫内病毒感染，导致脑白质丢失，并在脑脊液中产生高水平的干扰素α。许多AGS患者的这种疾病是由人类RNaseH2的三个亚单位中的任何一个突变引起的。我们对酿酒酵母同源酶的研究首次描述了真核细胞核糖核酸酶H2的三个亚基组成。在酿酒酵母中，任何编码RNaseH2三个亚基的基因的缺失都不会导致任何主要的表型，除非突变株与其他几个基因缺失中的任何一个结合在一起，这种情况被称为合成致死。我们目前正在研究人类和酿酒酵母RNaseH2的生化特性，特别是RNaseH2在DNA复制、修复和重组中的作用。我们的体外研究表明，在AGS患者中发现的大多数具有突变的蛋白质具有几乎正常的酶活性，但这些突变似乎可能影响与其他细胞蛋白质或成分的相互作用，从最近的结构分析来看，可能会发生一些亚单位相互作用的减弱，导致大脑比其他器官出现更严重的问题。 NIEHS最近的工作发现，酿酒酵母DNA聚合酶在复制过程中错误地将核糖核苷酸而不是脱氧核苷酸合并到DNA中。核糖核苷酸的去除依赖于RNaseH2的切割。

Research in the Section on Formation of RNA is directed toward understanding and utilization of processes involved in cellular DNA replication, the relationship of HIV replication to these cellular events, and how to use this information for therapeutic purposes. We are examining the formation and resolution of RNA/DNA hybrids occuring during DNA replication and/or transcription. Ribonucleases H are important enzymes participating in removal of the RNA of the RNA/DNA hybrids and are intimately related to DNA replication in cells and in HIV replication, during the conversion of the RNA genome of this virus to DNA. RNases H of cells and HIV share common enzymatic mechanisms of cleavage of RNA utilizing similar protein architectures. Drugs to alter levels of specific disease-related genes are being developed to take advantage of RNases H within the cell. Regulated expression of RNases H could enhance the efficacy of the drugs. Molecular genetic, biochemical, and mouse animal models are employed in these efforts. During transcription, RNA/DNA hybrids can form and are usually cleaved by RNase H. RNase H recognizes and specifically cleaves RNA of hybrids by several contacts to the RNA and interactions with the DNA strand using a two metal ion mechanism. We now know that the eukaryotic RNases H are more complex than the bacterial enzymes, and that the enzymatic activity of eukaryotic RNases H1 form a complex when binding to an RNA/DNA hybrid that enables the enzyme to be more efficient when degrading certain types of RNA/DNA hybrids. In collaboration with the groups at NCI-Frederick, we have been examined inhibitors of RNases H that could prove useful in HIV-AIDS therapy. From our earlier work showing that the mouse RNase H1 is critical for development, we know that it is important to have drugs that specifically target the HIV-AIDS RNase H not the cellular enzyme. Screening of potential drugs now includes use of the human RNase H1 isolated in our laboratory. Mouse RNase H1 is translated from a single messenger RNA to produce the same protein to reside in nuclei and in mitochondria. Production of the two forms depends on a translation process that we have found to be conserved in many eukaryotic organisms and in othe DNA transacting proteins targeted to both the nucleus and mitochondria. AGS is a syndrome is characterized as a severe neurological disorder that mimics in utero viral infection resulting in loss of white matter in the brain and producing high levels of interferon alpha in the cerebral spinal fluid. The disorder in many of the AGS patients results from mutations in any of the three subunits of human RNase H2. Our work on the homologous enzyme from Saccharomyces cerevisiae was the first describing the three subunit composition of eukaryotic RNases H2. Deletion of any genes encoding the three subunits of RNase H2 in S. cerevisiae does not result in any major phenotype unless the mutant is combined with any of several other gene deletions, a condition known as synthetic lethality. We are currently examining the biochemical properties of human and S. cerevisiae RNase H2, and, in particular, are examining the role of RNase H2 in DNA replication, repair and recombination. Our in vitro studies show that most of the proteins with mutations found in AGS patients have nearly normal enzymatic activity but it seems likely that these mutations affect interaction with other cellular proteins or components and from recent structural analysis, some weakening of the subunit interactions may occur leading to more severe problems in brain than in other organs. Recent work at NIEHS has found that S. cerevisiae DNA polymerases mistakenly incorporate ribonucleotides rather than deoxynucleotides into DNA during replication. Removal of the ribonucleotides depends on RNase H2 cleavage.