Factors Influencing Genetic Transcription Initiation And Termination

RNA形成部分的研究旨在了解RNA和DNA之间的相互作用，从而扰乱DNA，以及细胞如何对这些含有RNA的DNA做出反应。艾滋病病毒HIV使用RNA作为其基因组，当复制到DNA中时，RNA/DNA杂交物是需要RNaseH活性的中间产物，RNaseH是一种酶，在RNA被病毒DNA聚合酶(RT)复制后将其移除。RNaseH是RT的重要组成部分，可能成为治疗药物的靶点。RNA/DNA杂交体也存在于正常细胞中，在转录过程中偶尔会形成R环，在R环中，RNA取代一条DNA链，并与互补的DNA链形成双链。如果不解决，这些R-环可能会导致基因组不稳定。内源性核糖核酸酶H通常去除这些R-环。Aicardi Goutieres综合征(AGS)是一种罕见的自身免疫性疾病，具有严重的神经问题，可由人类RNaseH2缺陷引起。AGS模拟子宫病毒感染，包括大脑中白质的丢失和脑脊液中产生高水平的干扰素α。哺乳动物RNase H2可以降解RNA/DNA杂交物，但也可以识别双链DNA中的单个核苷酸并启动其去除。目前的建议表明，未能去除错误结合的核苷酸会导致DNA损伤，从而导致AGS。然而，当RNaseH2不存在时，RNA/DNA和DNA中的单核苷酸都会保留下来，这是到目前为止已经测试过的条件。因此，对于RNaseH2缺陷和AGS之间的关系仍然没有定论。我们已经使用酿酒酵母作为模式生物来研究RNaseH2两种活性所需的条件。有趣的是，我们有一个例子，其中RNaseH1或H2可以解析相同的R-环，而另一个例子中，R-环只被RNaseH2降解。为了更深入地了解它们对哺乳动物AGS相关突变的影响，我们培育了一只小鼠，它表达了在一些AGS患者中看到的RNaseH2，并一直在检查小鼠和小鼠组织的特性。 RNaseH1存在于哺乳动物细胞的线粒体(Mt)和细胞核中，是胚胎发育过程中产生mtDNA所必需的。它在核中的功能尚不完全清楚，但它对于解析与RNaseH2部分重叠作用的R环也可能是重要的。已经证明，在重复序列上形成R-环可以导致重组，从而导致扩展(复制)或缺失。当一个三联体重复，如CAG谷氨酰胺(Q)密码子扩展时，产生含有一种氨基酸的长延伸的蛋白质(CAG扩展的多Q)，就会出现几种人类疾病。通常情况下，这些蛋白质会聚集在一起，并由于这些复合体而影响细胞。在某些情况下，膨胀会直接影响蛋白质的功能。不难想象，移位的R-环DNA链可以通过多种方式与其互补的DNA链重新退火，从而导致修复和扩张或收缩。我们正在研究可能有这种三胞胎扩张的小鼠。 RNaseH1在线粒体DNA复制中的作用开始从与MRC剑桥大学线粒体单位的Ian Holt合作进行的一些研究中变得清晰起来。这些进展部分是通过使用RNaseH1产生缺陷的小鼠胚胎成纤维细胞取得的，这种缺陷导致细胞死亡和线粒体DNA中RNA/DNA的积累。 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。这两种形式的产生依赖于翻译过程，我们发现这种翻译过程在许多真核生物和其他针对细胞核和线粒体的DNA交易蛋白中是保守的。

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. If unresolved, these R-loops can 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 an RNase H2 seen in a few AGS patients and have been examining the properties of the mouse and mouse tissues. 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 mice that 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 of the Mitochondrial Unit at the MRC Cambridge. 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. 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 other DNA transacting proteins targeted to both the nucleus and mitochondria.