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。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RNase 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。目前的建议认为，这是未能去除掺入的核糖核苷酸导致DNA损伤引起AGS。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。为了解决这个问题，我们采用了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。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RNA酶H1或H2可以解析相同的R环，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AGS相关突变的影响，我们已经产生了表达在少数AGS患者中观察到的RNA酶H2突变形式的小鼠，RNA酶H2 G37 S，以及仅表达RNA酶H2 RED蛋白的小鼠。我们一直在研究老鼠和老鼠组织的特性。Rnaseh 2aG 37 S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。然而，通过基因失活该途径而丧失先天反应不允许形成可存活的幼崽。 我们发现只表达RNase H2 RED蛋白的小鼠与没有RNase H2的小鼠一样有缺陷，其中胚胎发育早在E9.5就有缺陷。这一发现与未能去除DNA中的核糖核苷酸（rNMPs）导致早期胚胎死亡一致。有趣的是，杂合子小鼠（Rnaseh 2 RED/G37 S）也是早期胚胎致死的。我们的研究结果表明，RNase H2 RED上的两种突变形式与RNase H2 G37 S竞争，允许在DNA中保留更多的rNMP。这通过杂合小鼠的DNA中rNMP的数量证实。仅具有RNase H2 G37 S的小鼠比正常小鼠含有更多的rNMP，但仍进行发育至出生。RNase H2 RED小鼠中的DNA损伤激活p53应答，而在RNase H2 G37 S小鼠中未观察到这种应答。这使我们得出结论，存在激活p53依赖性途径所需的rNMP的阈值，其在RED和G37 S小鼠中的数量之间。 与Aziz El Hage博士合作，Susana M. Cerritelli使用S.通过研究核糖核苷酸还原酶（RNR）的调节，酿酒酵母调节DNA中rNMPs的数量。rNTPs/dNTPs的比例是复制过程中掺入DNA的rNMP数量的主要贡献者。RNR可以通过基因操作变得更活跃或更不活跃。Cerritelli和El Hage改变了许多控制RNR的基因，证实了RNR相互作用途径，并在RNase H2如何响应RNR中的这些变化方面发现了重要的新发现。 这些关于DNA中rNMP丰度的发现是定义rNMP掺入DNA以及在人类疾病Aicardi-Goutires综合征的小鼠和酵母模型中未能将其去除的后果的主要贡献。

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.