Factors Influencing Genetic Transcription Initiation And Termination

解释 RNA形成部分的研究旨在了解RNA和DNA之间干扰DNA的相互作用以及细胞如何对这些含RNA的DNA做出反应。艾滋病病毒，HIV，使用RNA作为其基因组，当复制成DNA时，RNA/DNA杂交体是需要RNase H活性的中间体，RNase H是一种在RNA被病毒DNA聚合酶（逆转录酶或RT）复制后将其去除的酶。RNase H是RT的重要组成部分，可能成为治疗药物的靶点。RNA/DNA杂合体也存在于正常细胞中，偶尔在转录过程中形成，产生R环，其中RNA取代DNA的一条链并与互补DNA链形成双链体。此外，重组蛋白可以通过置换一条DNA链，同时将互补RNA退火至另一条DNA链来形成RNA/DNA杂交体。如果未解决，这些R环会导致基因组不稳定。内源性RNA酶H通常去除这些R环。古铁雷斯综合征（AGS）是一种罕见的自身免疫性疾病，具有严重的神经系统问题，可由人类RNA酶H2缺陷引起。AGS模拟子宫内病毒感染，包括脑中白色物质的损失和在脑脊液中产生高水平的干扰素α。哺乳动物RNA酶H2可以降解RNA/DNA杂合体，但也可以识别双链体DNA中的单个核糖核苷酸并启动其去除。目前的研究认为，AGS的发生是由于未能清除错配的核糖核苷酸导致DNA损伤所致。然而，当RNase H2不存在时，RNA/DNA和DNA中的单个核糖核苷酸仍然存在;迄今为止测试的条件。因此，对于RNase H2缺陷和AGS之间的关系，目前还没有定论。我们已经采用酿酒酵母作为模式生物，以检查RNase H2的两种活性所需的条件。有趣的是，我们有一个例子，其中RNA酶H1或H2可以解析相同的R环，而另一个例子中R环只被RNA酶H2降解。为了更深入地了解其对哺乳动物中与AGS相关的突变的影响，我们已经产生了一种表达在一些AGS患者中观察到的RNA酶H2突变形式的小鼠，并一直在检查小鼠和小鼠组织的性质。突变纯合子小鼠出生时死亡或出生后不久死亡。DNA损伤导致cGAS/Sting先天免疫应答途径的诱导。 核糖核酸酶H1存在于哺乳动物细胞的线粒体（mt）和细胞核中，在胚胎发育过程中需要产生mtDNA。它在细胞核中的功能尚未完全了解，但它可能对于解析具有与RNase H2部分重叠作用的R环也很重要。已经表明，重复序列处的R环形成可导致重组，从而导致扩增（复制）或缺失。当三联体重复序列如CAG谷氨酰胺（Q）密码子扩增产生含有一个氨基酸的长片段的蛋白质（polyQ用于CAG扩增）时，出现几种人类疾病。通常这些蛋白质会聚集，并由于这些复合物而影响细胞。在某些情况下，扩增对具有扩增的蛋白质的功能具有直接影响。很容易想象，R环的置换DNA链可以以多种方式重新退火到其互补DNA链，从而导致修复和扩张或收缩。我们正在研究在我们的RNase H1敲除株中出现的共济失调小鼠可能具有这种三联体扩增。 核糖核酸酶H1在线粒体DNA复制中的作用开始变得清晰，这是与西班牙Gipuzkoa的Biodonostia健康研究所的Ian Holt合作进行的一些研究。这些进展部分是通过使用小鼠胚胎成纤维细胞来实现的，这些成纤维细胞具有导致细胞死亡和mtDNA中RNA/DNA积累的RNA酶H1产生缺陷。 Holts实验室开发的模型表明，RNA是mtDNA复制的主要中间体，可能通过类似于重组相关蛋白介导的RNA/DNA杂交形成的机制。 最近，我们一直在研究导致线粒体疾病的RNASEH 1人类基因的缺陷。酶结构的改变是相当温和的，但会给患者带来严重的致残问题。

Explanation Research in the Section on Formation of RNA is directed toward understanding the interaction between RNA and DNA that perturb the DNA and how the cell responds to these RNA-containing DNAs. The AIDS virus, HIV, employs RNA as its genome and when copied into DNA RNA/DNA hybrids are intermediates that require RNase H activity, an enzyme that removes the RNA after it is copied by the viral DNA polymerase (Reverse Transcriptase or RT). RNase H is an essential part of RT and could be a target for therapeutic drugs. RNA/DNA hybrids are also present in normal cells, occasionally forming during transcription producing R-loops in which the RNA displaces one strand of DNA and forms a duplex with the complementary DNA strand. In addition, recombination proteins can form RNA/DNA hybrids by displacing one DNA strand while annealing complementary RNA to the other DNA strand. If unresolved, these R-loops lead to genome instability. The endogenous RNases H usually remove these R-loops. Aicardi Goutieres Syndrome (AGS) is a rare autoimmune disorder with severe neurological problems that can be caused by defects in human RNase H2. AGS mimics in utero viral infection including loss of white matter in the brain and producing high levels of interferon alpha in the cerebral spinal fluid. Mammalian RNases H2 can degrade RNA/DNA hybrids but can also recognize a single ribonucleotide in duplex DNA and initiates its removal. Current proposals suggest it is the failure to remove the missincorporated ribonucleotides results in DNA damage causing AGS. However, both RNA/DNA and single ribonucleotides in DNA remain when RNase H2 is not present; conditions tested so far. Thus, the jury is still out on the relationship between RNase H2 defects and AGS. We have been employed Saccharomyces cerevisiae as a model organism to examine conditions under which the two activities of RNase H2 are required. Interestingly, we have example where either RNase H1 or H2 can resolve the same R-loops and another in which R-loops are only degraded by RNase H2. To gain more insight into the effects of the same into AGS-related mutations in mammals, we have generated a mouse which expresses a mutant form of RNase H2 seen in a few AGS patients and have been examining the properties of the mouse and mouse tissues. The mice homozygous for the mutation are born dead or or die soon after birth. DNA damage leads to induction of the cGAS/Sting innate immune response pathway. RNase H1 is present in mitochondria (mt) and nuclei of mammalian cells and is required during embryonic development to generate mtDNA. Its function in the nucleus is not completely understood but it too is likely important for resolving R-loops with partial overlapping action with RNase H2. It has been shown that R-loop formation at repetitive sequences can lead to recombination that result in expansion (duplication) or deletion. Several human disorders arise when a triplet repeat such as CAG glutamine (Q) codon expand producing proteins containing long stretches of one amino acid (polyQ for CAG expansion). Often these proteins become aggregated and can affect the cell due to these complexes. In some cases, the expansion has direct affects on the function of the protein with the expansion. It is easy to imagine that the displaced DNA strand of R-loops can reanneal to its complementary DNA strand in multiple ways leading to repair and expansion or contraction. We are examining ataxic mice that arose in our RNase H1 knockout strain may have such triplet expansions. The role of RNase H1 in mtDNA replication is beginning to become clear from some of the studies carried out in collaboration with Ian Holt at Biodonostia Health Research Institute, Gipuzkoa, Spain. These advances have been made in part by using mouse embryo fibroblasts with defects in RNase H1 production that lead to cell death and accumulation of RNA/DNA in mtDNA. The model developed in Holts lab suggests RNA is a major intermediate in mtDNA replication, possibly by a mechanism similar to that described for RNA/DNA hybrid formation mediated by a recombination-related protein. Recently, we have been examining defects in the RNASEH1 human gene which leads to a mitochondrial disorder. The modification of the structure of the enzyme is quite modest but creates a severe disabling problem for the patient.