R-loop Processing Research Articles

RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks

Although accumulation of DNA damage and genomic instability in resting cells can cause neurodegenerative disorders, our understanding of how transcription produces DNA double-strand breaks (DSBs) islimited. Transcription-blocking topoisomerase I cleavage complexes (TOP1ccs) are frequent events that prime DSB production in non-replicating cells. Here, we report a mechanism of their formation by showing that they arise from two nearby single-strand breaks (SSBs) on opposing DNA strands: one SSB from the removal of transcription-blocking TOP1ccs by the TDP1 pathway and the other from the cleavage of R-loops by endonucleases, including XPF, XPG, and FEN1. Genetic defects in TOP1cc removal (TDP1, PNKP, and XRCC1) or in the resolution of R-loops (SETX) enhance DSB formation and prevent their repair. Such deficiencies cause neurological disorders. Owing to the high frequency of TOP1cc trapping and the widespread distribution of R-loops, these persistent transcriptional DSBs could accumulate over time in neuronal cells, contributing to the neurodegenerative diseases.

Trypanosoma brucei ribonuclease H2A is an essential R-loop processing enzyme whose loss causes DNA damage during transcription initiation and antigenic variation.

Ribonucleotides represent a threat to DNA genome stability and transmission. Two types of Ribonuclease H (RNase H) excise ribonucleotides when they form part of the DNA strand, or hydrolyse RNA when it base-pairs with DNA in structures termed R-loops. Loss of either RNase H is lethal in mammals, whereas yeast survives the absence of both enzymes. RNase H1 loss is tolerated by the parasite Trypanosoma brucei but no work has examined the function of RNase H2. Here we show that loss of T. brucei RNase H2 (TbRH2A) leads to growth and cell cycle arrest that is concomitant with accumulation of nuclear damage at sites of RNA polymerase (Pol) II transcription initiation, revealing a novel and critical role for RNase H2. Differential gene expression analysis reveals limited overall changes in RNA levels for RNA Pol II genes after TbRH2A loss, but increased perturbation of nucleotide metabolic genes. Finally, we show that TbRH2A loss causes R-loop and DNA damage accumulation in telomeric RNA Pol I transcription sites, also leading to altered gene expression. Thus, we demonstrate separation of function between two nuclear T. brucei RNase H enzymes during RNA Pol II transcription, but overlap in function during RNA Pol I-mediated gene expression during host immune evasion.

RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Human mitochondrial DNA (mtDNA) replication is first initiated at the origin of H-strand replication. The initiation depends on RNA primers generated by transcription from an upstream promoter (LSP). Here we reconstitute this process in vitro using purified transcription and replication factors. The majority of all transcription events from LSP are prematurely terminated after ~120 nucleotides, forming stable R-loops. These nascent R-loops cannot directly prime mtDNA synthesis, but must first be processed by RNase H1 to generate 3′-ends that can be used by DNA polymerase γ to initiate DNA synthesis. Our findings are consistent with recent studies of a knockout mouse model, which demonstrated that RNase H1 is required for R-loop processing and mtDNA maintenance in vivo. Both R-loop formation and DNA replication initiation are stimulated by the mitochondrial single-stranded DNA binding protein. In an RNase H1 deficient patient cell line, the precise initiation of mtDNA replication is lost and DNA synthesis is initiated from multiple sites throughout the mitochondrial control region. In combination with previously published in vivo data, the findings presented here suggest a model, in which R-loop processing by RNase H1 directs origin-specific initiation of DNA replication in human mitochondria.

Sae2/CtIP prevents R-loop accumulation in eukaryotic cells.

The Sae2/CtIP protein is required for efficient processing of DNA double-strand breaks that initiate homologous recombination in eukaryotic cells. Sae2/CtIP is also important for survival of single-stranded Top1-induced lesions and CtIP is known to associate directly with transcription-associated complexes in mammalian cells. Here we investigate the role of Sae2/CtIP at single-strand lesions in budding yeast and in human cells and find that depletion of Sae2/CtIP promotes the accumulation of stalled RNA polymerase and RNA-DNA hybrids at sites of highly expressed genes. Overexpression of the RNA-DNA helicase Senataxin suppresses DNA damage sensitivity and R-loop accumulation in Sae2/CtIP-deficient cells, and a catalytic mutant of CtIP fails to complement this sensitivity, indicating a role for CtIP nuclease activity in the repair process. Based on this evidence, we propose that R-loop processing by 5' flap endonucleases is a necessary step in the stabilization and removal of nascent R-loop initiating structures in eukaryotic cells.

RAD52 Promotes Transcription-Associated Homologous Recombination Repair

Abstract RAD52-mediated processing of R-loops initiates high-fidelity DNA repair in actively transcribed regions.

Human Rad52 Promotes XPG-Mediated R-loop Processing to Initiate Transcription-Associated Homologous Recombination Repair

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.

Aquarius is required for proper CtIP expression and homologous recombination repair

Accumulating evidence indicates that transcription is closely related to DNA damage formation and that the loss of RNA biogenesis factors causes genome instability. However, whether such factors are involved in DNA damage responses remains unclear. We focus here on the RNA helicase Aquarius (AQR), a known R-loop processing factor, and show that its depletion in human cells results in the accumulation of DNA damage during S phase, mediated by R-loop formation. We investigated the involvement of Aquarius in DNA damage responses and found that AQR knockdown decreased DNA damage-induced foci formation of Rad51 and replication protein A, suggesting that Aquarius contributes to homologous recombination (HR)-mediated repair of DNA double-strand breaks (DSBs). Interestingly, the protein level of CtIP, a DSB processing factor, was decreased in AQR-knockdown cells. Exogenous expression of Aquarius partially restored CtIP protein level; however, CtIP overproduction did not rescue defective HR in AQR-knockdown cells. In accordance with these data, Aquarius depletion sensitized cells to genotoxic agents. We propose that Aquarius contributes to the maintenance of genomic stability via regulation of HR by CtIP-dependent and -independent pathways.

Replication-Transcription Conflicts Generate R-Loops that Orchestrate Bacterial Stress Survival and Pathogenesis

Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis. We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B.subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis.

Abstract 1430: The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing

Abstract (Objective)Recent whole-exome sequencing studies of malignancies have detected recurrent somatic mutations in U2 snRNP components of the spliceosome. These factors have also been listed as novel players of DNA damage response in several genome wide screens and proteome analysis for DDR genes. Although accumulating evidences have implied that the spliceosome plays an important role in genome stability and is an emerging hallmark of carcinogenic pathways, its precise role in genome stability still remains ambiguous. The aim of this study is to clarify the functions of U2 snRNP splicing factors, especially SNRPA1 (Small Nuclear Ribonucleoprotein Polypeptide A1) in DDR pathway. (Methods)SNAPA1 and other U2 snRNP splicing factors were identified as HR repair genes by genome-wide screens based on homologous recombination and RAD51 immunofoci formation. Each splicing factor was depleted by siRNA knockdown in USOS cells. The functions of SNRPA1 in DNA repair were analyzed by HR assay (DR-GFP assay), immunofluorescence, real-time laser micro-irradiation and comet assay (single cell gel electrophoresis). (Results)HR assay showed strong HR deficiencies in splicing factor's depleted cells. In these cells, accumulation of BRCA1 and Rad51, major HR factors, to DNA damage sites were severely impaired. Especially, live cell imaging showed recruitment of SNRPA1 to laser induced DNA damage sites and unveiled its direct involvement to DNA damage repair. Comet assay also showed that depletion of SNRPA1 markedly caused DNA damage with the tail of broken DNA fragments. This DNA damage was R-loop (DNA-RNA hybrid) mediated DNA damage and rescued by overexpression of RNAseH1. (Conclusion) Here we unveiled two distinct pathways how spliceosome U2 snRNP factors contribute to genome stability. The main function is indirect, through transcription, to maintain the protein levels of essential repair factors and contribute to homologous recombination repair. Our data suggest that depletion of splicing factors can be one mechanism for HR deficiency (i.e. BRCAness). In addition real-time laser microirradiation analysis identified the rapid recruitment of SNRPA1 to DNA damage sites. Intensive functional analysis of SNRPA1 unveiled the other, more immediate and direct effect to process R-loop structure, deleterious transcriptional by-products for the genome, at sites of on-going transcription. Citation Format: Michihiro Tanikawa. The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1430. doi:10.1158/1538-7445.AM2017-1430

The spliceosome U2 snRNP factors promote genome stability through distinct mechanisms; transcription of repair factors and R-loop processing.

Recent whole-exome sequencing of malignancies have detected recurrent somatic mutations in U2 small nuclear ribonucleoprotein complex (snRNP) components of the spliceosome. These factors have also been identified as novel players in the DNA-damage response (DDR) in several genome-wide screens and proteomic analysis. Although accumulating evidence implies that the spliceosome has an important role in genome stability and is an emerging hallmark of cancer, its precise role in DNA repair still remains elusive. Here we identify two distinct mechanisms of how spliceosome U2 snRNP factors contribute to genome stability. We show that the spliceosome maintains protein levels of essential repair factors, thus contributing to homologous recombination repair. In addition, real-time laser microirradiation analysis identified rapid recruitment of the U2 snRNP factor SNRPA1 to DNA-damage sites. Functional analysis of SNRPA1 revealed a more immediate and direct role in preventing R-loop-induced DNA damage. Our present study implies a complex interrelation between transcription, mRNA splicing and the DDR. Cells require rapid spatio-temporal coordination of these chromatin transactions to cope with various forms of genotoxic stress.

Viable RNaseH1 knockout mice show RNaseH1 is essential for R loop processing, mitochondrial and liver function.

Viable constitutive and tamoxifen inducible liver-specific RNase H1 knockout mice that expressed no RNase H1 activity in hepatocytes showed increased R-loop levels and reduced mitochondrial encoded DNA and mRNA levels, suggesting impaired mitochondrial R-loop processing, transcription and mitochondrial DNA replication. These changes resulted in mitochondrial dysfunction with marked changes in mitochondrial fusion, fission, morphology and transcriptional changes reflective of mitochondrial damage and stress. Liver degeneration ensued, as indicated by apoptosis, fibrosis and increased transaminase levels. Antisense oligonucleotides (ASOs) designed to serve as substrates for RNase H1 were inactive in the hepatocytes from the RNase H1 knockout mice and in vivo, demonstrating that RNase H1 is necessary for the activity of DNA-like ASOs. During liver regeneration, a clone of hepatocytes that expressed RNase H1 developed and partially restored mitochondrial and liver function.

Processing of double-R-loops in (CAG)·(CTG) and C9orf72 (GGGGCC)·(GGCCCC) repeats causes instability.

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.

R-loop Processing by BRCA2 Is Required for Genomic Stability

Abstract BRCA2 interacts with TREX-2 subunits and is required to prevent R-loop–associated DNA damage.

On the viability of Escherichia colicells lacking DNA topoisomerase I

BackgroundManipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I.ResultsIn this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG.ConclusionsOur data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.

Initiation of Mitochondrial DNA Replication by Transcription and R-loop Processing

The mitochondrial genome of eukaryotic cells is maintained by a mechanism distinct from that employed in the nucleus. Mitochondrial DNA replication at the leading-strand origin is coupled to transcription through the formation of an RNA-DNA hybrid known as an R-loop. In vivo and in vitro evidence has implicated an RNA processing enzyme, RNase MRP, in primer maturation. In our investigation of mammalian RNase MRP, we have analyzed its specific endoribonuclease activity on model R-loops. We demonstrate here that human RNase MRP cleaves this distinctly configured substrate at virtually all of the major DNA replication sites previously mapped in vivo. We further show that the processed RNA products remain stably base-paired to the template DNA strand and are functional for initiating DNA synthesis on a closed circular plasmid. Thus, in vitro initiation of leading-strand mtDNA synthesis requires only the actions of RNA polymerase and RNase MRP for the generation of replication primers.