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。此外，重组蛋白可以通过置换一条DNA链，同时将互补的RNA退火到另一条DNA链上，从而形成RNA/DNA杂交物。如果不解决，这些R-环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 Goutires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，未能移除被结合的核糖核苷酸会导致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；到目前为止，条件已经过测试。为了解决这个问题，我们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。我们称这种酶为RNase H2 RED(RNA切除缺陷)。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。 为了更好地了解它们对哺乳动物AGS相关突变的影响，我们培育了一只表达在少数AGS患者中看到的RNaseH2突变形式的小鼠，RNaseH2G37S，以及一只只表达RNaseH2RED蛋白的小鼠。我们一直在检查小鼠和小鼠组织的特性。RNaseh2aG37S突变纯合的小鼠出生时死亡或出生后不久死亡。DNA损伤导致cGAS/Sting先天免疫反应通路的诱导。然而，通过基因失活这一途径而失去先天反应，并不能形成可存活的幼崽。 我们发现，只表达RNaseH2RED蛋白的小鼠和没有RNase H2的小鼠一样有缺陷，后者的胚胎发育早在E9.5就有缺陷。这一发现与未能去除DNA中的核糖核苷酸(RNMP)会导致早期胚胎死亡是一致的。有趣的是，杂合子(RNaseh2RED/G37S)的小鼠也是早期胚胎致死的。我们的结果表明，RNaseH2RED上的两种突变形式与RNaseH2G37S竞争，从而允许更多的rNMPs保留在DNA中。杂合子小鼠DNA中rNMPs的数量证实了这一点。仅携带RNaseH2G37S的小鼠比正常小鼠含有更多的rNMPs，但仍在发育到出生阶段。RNaseH2RED小鼠的DNA损伤激活了P53反应，而RNaseH2G37S小鼠则没有观察到这种反应。这导致我们得出结论，激活P53依赖的途径需要一个rNMPs的阈值，这个阈值介于红色和G37S小鼠的数量之间。 在与Aziz El Hage博士的合作中，Susana M.Cerritelli博士利用酿酒酵母通过研究核糖核苷酸还原酶(RNR)的调节来调节DNA中rNMP的数量。RNTP/dNTP的比率是复制过程中纳入DNA的rNMP数量的主要贡献者。RnR可以通过基因操作而变得更活跃或更不活跃。Cerritelli和El Hage已经改变了许多控制RNR的基因，确认了RNR的相互作用途径，并在RNase H2如何响应这些变化方面发现了重要的新发现。 这些关于DNA中rNMPs丰度的发现是定义rNMPs整合到DNA中的主要贡献，以及在人类疾病Aicardi-Goutires综合征的特征的小鼠和酵母模型中未能去除rNMPs的后果。

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 Goutires 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 incorporated 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. To address this question, we have employed Saccharomyces cerevisiae as a model organism to examine conditions under which the two activities of RNase H2 are required. We call this enzyme RNase H2RED (RNA Excision Defective). Interestingly, we have an 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, RNase H2G37S, and a mouse expressing only the RNase H2RED protein. We have been examining the properties of the mouse and mouse tissues. The mice homozygous for the Rnaseh2aG37S mutation are born dead or die soon after birth. DNA damage leads to induction of the cGAS/Sting innate immune response pathway. However, loss of an innate response by genetically inactivating that pathway does not permit formation of viable pups. We have found mice expressing only the RNase H2RED protein are as defective as mice with no RNase H2, where embryonic development is defective as early as E9.5. This finding is consistent with failure to remove ribonucleotides (rNMPs) in DNA causes early embryonic lethality. Interestingly, mice that are heterozygous (Rnaseh2RED/G37S) are also early embryonic lethal. Our results indicate the two mutant forms on RNase H2RED competes with RNase H2G37S allowing retention of more rNMPs in DNA. This is confirmed by the numbers of rNMPs in DNA for the heterozygous mice. The mice with only RNase H2G37S contain more rNMPs than normal mice but still progress development to birth. DNA damage in RNase H2RED mice activates p53 response whereas such a response is not observed in RNase H2G37S mice. This leads us to conclude that there is a threshold of rNMPs required to activate the p53-dependent pathway which is between the number in the RED and G37S mice. In collaboration with Dr. Aziz El Hage, Dr. Susana M. Cerritelli has used S. cerevisiae to modulate the number of rNMPs in DNA by studying the regulation of ribonucleotide reductase (RNR). The ratio of rNTPs/dNTPs is a major contributor to the number of rNMPs incorporated into DNA during replication. RNR can be genetically manipulated to be more and less active. Cerritelli and El Hage have altered many genes controlling RNR and have confirmed the RNR interactive-pathway as well as found important new findings in how RNase H2 respond to these change in RNR. These findings about abundance of rNMPs in DNA are major contributions in defining the incorporation of rNMPs into DNA and the consequences of failure to remove them in both mouse and yeast models of properties of the human disorder, Aicardi-Goutires Syndrome.