Factors Influencing Genetic Transcription Initiation And Termination

总结 我们的研究重点是核糖核酸酶H，这是维持基因组稳定性所必需的酶。核糖核酸酶H识别RNA/DNA杂合体并消化RNA链。RNA/DNA杂交体的形成发生在转录、DNA复制和HIV-AIDS病毒或内源性逆转录病毒元件复制期间。已知两种类型的细胞RNA酶H。RNA酶H1和H2都可以处理RNA/DNA杂合体和R环，这是在转录过程中当从RNA聚合酶中挤出的mRNA与模板DNA杂交时形成的三股结构，留下单链形式的非模板链。此外，RNase H2可以在复制过程中切割嵌入基因组DNA中的单个核糖核苷单磷酸（rNMP），通过无错误的核糖核苷酸切除修复（RER）途径启动其去除。我们已经描述了RNA酶H1和H2的基本结构，现在的目标是研究它们在体内的影响。1型突变由于不能扩增线粒体DNA而导致小鼠胚胎发育停滞。具有突变的RNA酶H1的人类患者表现出具有丝裂肌病患者的典型症状。HIV-AIDS病毒的复制需要病毒编码的RNase H，这是产生感染性病毒颗粒所必需的，使其成为阻断病毒复制的药物的有用靶点。1型和HIV-AIDS酶在结构和作用机制上相似，因此必须检查任何潜在药物对细胞1型RNA酶H的影响。2型核糖核酸酶H的突变可导致严重的神经系统综合征。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RNA酶H2的亚基。了解这种RNase H与AGS的关系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的作用，以获得基本信息，以及这些酶如何与各种疾病和病症的原因相关。 作为与爱丁堡大学的Aziz El Hage和大卫托勒维合作的一部分，我们正在酵母中研究在没有RNase H的情况下，调节DNA中rNMP的掺入如何影响细胞活力。我们分两步高度增加DNA中rNMP的密度。首先，通过消耗核糖核苷酸还原酶（RNR），其是核糖核苷酸转化为脱氧核糖核苷酸的限制酶。在较低的dNTP/rNTP比率下，复制型DNA聚合酶对rNTP的区分性较低。我们观察到，在没有RNase H1和H2的情况下，细胞不能在DNA中过量的rNMP中存活。在这种情况下，细胞死亡是由DNA和RNA/DNA杂合体中的rNMP引起的累积的和不可修复的DNA损伤诱导的。第二，我们通过使用对核糖核苷酸具有较低严格性的复制型DNA聚合酶，除了耗尽RNR之外，还加剧了DNA中的rNMP掺入。在这些条件下，RNA酶H2以拓扑异构酶1依赖性方式变得必不可少。在不存在RNase H2的情况下，DNA拓扑异构酶1（一种分解扭转应力所需的酶）以高度致突变的方式处理单个基因组核糖核苷酸，导致致死性。我们的结论是，在酵母中，在哺乳动物中一样，有一个阈值的耐受性的积累基因组核糖核苷酸在RNase H2的情况下，高于此有细胞死亡。这项工作于2020年3月发表在《核酸研究》上，也是刚刚发表在《当代遗传学》（2020年9月）上的特邀评论的主题。 在2019年，罗伯特·克劳奇和苏珊娜·塞里泰利应邀编辑了DNA修复特刊，名为RNases H：维持基因组完整性的多重作用。这期特刊包含了10篇由该领域领导人撰写的文章，包括克劳奇和切里泰利的介绍和评论文章。该问题于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.