Factors Influencing Genetic Transcription Initiation And Termination

点击翻译按钮获取中文摘要

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.