Factors Influencing Genetic Transcription Initiation And Termination

摘要 我们的研究重点是核糖核酸酶H，这是维持基因组稳定所必需的酶。核糖核酸酶H识别RNA/DNA杂交物并消化RNA链。RNA/DNA杂交物的形成发生在HIV-AIDS病毒或内源性逆转录病毒元件的转录、DNA复制和复制过程中。已知两种类型的细胞核糖核酸酶H。RNaseH1和H2都能处理RNA/DNA杂交物和R-环，这是从RNA聚合酶挤出的mRNA与模板DNA杂交时在转录过程中形成的三条链结构，使非模板链以单链形式存在。此外，RNase H2可以在复制过程中切割嵌入基因组DNA中的单个核糖核苷一磷酸(RNMP)，通过无错误的核苷酸切除修复(RER)途径将其移除。我们已经描述了RNaseH1和H2的基本结构，现在的目标是研究它们在体内的影响。由于未能扩增线粒体DNA，1型突变会导致小鼠胚胎发育停滞。人类RNaseH1突变患者表现出典型的有丝分裂肌病患者的症状。复制艾滋病毒-艾滋病病毒需要病毒编码的RNaseH，这是产生具有感染性的病毒颗粒所必需的，使其成为药物阻止病毒复制的有用靶点。1型和HIV-AIDS酶在结构和作用机制上相似，因此有必要研究任何潜在的药物对细胞1型RNaseH的影响。Aicardi Goutires综合征(AGS)是一种严重的人类疾病，由五个基因突变引起，其中三个基因编码RNaseH2亚单位。了解这个RNaseH是如何与AGS相关的是我们的主要努力之一。 我们目前正在使用酵母和小鼠模型来深入了解RNases H的基本信息以及这些酶是如何与各种疾病和紊乱的原因相关的。 作为与爱丁堡大学的Aziz El Hage和David Tollervey合作的一部分，我们正在酵母中研究在没有RNase H的情况下，调节DNA中rNMPs的并入如何影响细胞活力。我们分两步极大地提高了DNA中rNMPs的密度。首先，通过消耗核糖核苷酸还原酶(RNR)，这是核糖核苷酸转化为脱氧核糖核苷酸的限制酶。当dNTP/rNTP比率较低时，复制型DNA聚合酶对rNTP的鉴别力较低。我们观察到，在没有RNaseH1和H2的情况下，细胞不能在DNA中过量的rNMPs中存活。在这种情况下，细胞死亡是由rNMPs在DNA和RNA/DNA杂交体中造成的累积性和不可修复的DNA损伤引起的。其次，除了消耗RNR外，我们还使用了对核糖核苷酸具有较低严格性的复制型DNA聚合酶，从而加剧了rNMP在DNA中的掺入。在这些条件下，RNaseH2以拓扑异构酶1依赖的方式变得必不可少。在没有RNaseH2的情况下，DNA拓扑异构酶1(一种分解扭转应力所需的酶)以高度突变的方式处理单个基因组核苷酸，导致致命性。我们的结论是，在酵母中，就像在哺乳动物中一样，在缺乏RNaseH2的情况下，对基因组核苷酸的积累有一个耐受阈值，超过这个阈值就会出现细胞死亡。这项研究发表在2020年3月的《核酸研究》上，也是刚刚在《当代遗传学》上发表的一篇特邀评论的主题(2020年9月)。 在2019年，罗伯特·克劳奇和苏珊娜·切里泰利应邀编辑了一期DNA修复特刊，名为RNase H：Multiple Role in Maining Genome Integrity。特刊收录了该领域领导人撰写的10篇文章，其中包括克劳奇和切里泰利撰写的一篇导言和一篇评论文章。这期杂志于2019年12月出版，封面由我们的posbac Yasmeen Ajaj设计。

Summary Our studies focus on Ribonucleases H, enzymes which are essential to maintain genome stability. Ribonucleases H recognize RNA/DNA hybrids and digest the RNA strand. Formation of RNA/DNA hybrids occurs during transcription, DNA replication, and during replication of the HIV-AIDS virus or endogenous retroviral elements. Two types of cellular RNases H are known. Both RNase H1 and H2 can process RNA/DNA hybrids and R-loops, which are three strands structures formed during transcription when the mRNA extruding from the RNA Polymerase hybridized to the template DNA leaving the non-template strand in single stranded form. In addition, RNase H2 can incise at single ribonucleoside-monophosphates (rNMPs) embedded in genomic DNA during replication, initiating their removal by the error-free, ribonucleotide excision repair (RER) pathway. We have described the basic structure of both RNase H1 and H2 and are now aiming to study their impact in vivo. Mutations in type 1 lead to arrest of embryonic development in mice due to a failure to amplify mitochondrial DNA. Human patients with mutated RNase H1 exhibit typical symptoms of patients with mitomyopathies. Replication of the HIV-AIDS virus requires a virally encode RNase H that is necessary for production of infectious viral particles, making it a useful target for drugs to block replication of the virus. Type 1 and the HIV-AIDS enzymes are similar in structure and mechanism of action, making imperative to examine effects of any potential drugs on the cellular type 1 RNase H. Mutations in type 2 RNase H can lead to a serious neurological syndrome. Aicardi Goutires Syndrome (AGS) is a severe human disorder caused by mutations in five genes, three of which encode subunits of RNase H2. Understanding how this RNase H is related to AGS is one of our major efforts. We are currently using yeast and mouse models to gain insight into roles of RNases H for basic information and how these enzymes are related to the cause of various diseases and disorders. As part of a collaboration with Aziz El Hage and David Tollervey from the University of Edinburgh, we are studying in yeast how modulating the incorporation of rNMPs in DNA affects cell viability in the absence of RNases H. We highly increased the density of rNMPs in DNA in two steps. First, by depleting ribonucleotide reductase (RNR), which is the limiting enzyme in the conversion of ribonucleotides to deoxyribonucleotides. With lower dNTP/rNTP ratios, replicative DNA polymerases are less discriminative against rNTPs. We observed that cells could not survived the excess of rNMPs in DNA in the absence of RNase H1 and H2. In this situation, cell death is induced by accumulative and irreparable DNA damage caused by rNMPs in DNA and RNA/DNA hybrids. Second, we exacerbated rNMP incorporation in DNA by, in addition of depleting RNR, using replicative DNA polymerases that have lower stringency against ribonucleotides. In these conditions, RNase H2 becomes essential in a Topoisomerase 1-dependent manner. In absence of RNase H2, DNA Topoisomerase 1 (an enzyme that is required to resolve torsional stress) processes single genomic ribonucleotides in a highly mutagenic manner, leading to lethality. We conclude that in yeast, like in mammals, there is a threshold of tolerance for the accumulation of genomic ribonucleotides in absence of RNase H2, above which there is cell death. This work was published in Nucleic Acid Research on March 2020 and was the topic of an invited review just published (September 2020) in Current Genetics. During 2019, Robert Crouch and Susana Cerritelli edited by invitation a Special Issue for DNA Repair called RNases H: Multiple Roles in Maintaining Genome Integrity. The Special Issue contained 10 articles written by leaders in the field, including an introduction and a review article by Crouch and Cerritelli. The issue was published in December 2019 and the cover was designed by our posbac,Yasmeen Ajaj.