RNase H eliminates R‐loops that disrupt DNA replication but is nonessential for efficient DSB repair

In Saccharomyces cerevisiae, genome stability depends on RNases H1 and H2, which remove ribonucleotides from DNA and eliminate RNA–DNA hybrids (R‐loops). In Schizosaccharomyces pombe, RNase H enzymes were reported to process RNA–DNA hybrids produced at a double‐strand break (DSB) generated by I‐PpoI meganuclease. However, it is unclear if RNase H is generally required for efficient DSB repair in fission yeast, or whether it has other genome protection roles. Here, we show that S. pombe rnh1? rnh201? cells, which lack the RNase H enzymes, accumulate R‐loops and activate DNA damage checkpoints. Their viability requires critical DSB repair proteins and Mus81, which resolves DNA junctions formed during repair of broken replication forks. “Dirty” DSBs generated by ionizing radiation, as well as a “clean” DSB at a broken replication fork, are efficiently repaired in the absence of RNase H. RNA–DNA hybrids are not detected at a reparable DSB formed by fork collapse. We conclude that unprocessed R‐loops collapse replication forks in rnh1? rnh201? cells, but RNase H is not generally required for efficient DSB repair.     

细菌中RNase HI介导RNA降解的研究进展

在细菌细胞中,为了维持基因组稳定和正常的生命活动,RNase HI通常以降解RNA/DNA杂合链中RNA的方式来防止复制中引物的积累以及转录中R环的形成。RNase HI对底物的识别主要依赖于DNA与RNA结合槽,对底物的催化主要依赖于DEDD基序和位于活性位点附近柔性环中的一个组氨酸。以Mg²⁺为代表的金属离子在催化过程中发挥了至关重要的作用。杂交双链中ssDNA突出部分的类型决定了RNase HI的作用模式：在没有突出或在ssDNA的5′端存在突出部分的情况下,RNase HI作为一种非序列特异性核酸内切酶随机地降解RNA;当ssDNA的3′端存在突出部分时,RNase HI依靠5′核酸外切酶活性对RNA进行连续切割。RNase HI、Rep、DinG和UvrD通过与单链DNA结合蛋白(single-stranded DNA-binding protein, SSB)的C端尾部的6个残基相互作用被招募到复制叉附近,并可能以协作的方式解决复制-转录冲突。RNaseHI的缺失或活性降低将引起DNA结构不稳定、基因突变、转录装置回溯和复制不协调等一系列有害后果。RN...     

Topoisomerase I‐mediated cleavage at unrepaired ribonucleotides generates DNA double‐strand breaks

Ribonuclease activity of topoisomerase I (Top1) causes DNA nicks bearing 2′,3′‐cyclic phosphates at ribonucleotide sites. Here, we provide genetic and biochemical evidence that DNA double‐strand breaks (DSBs) can be directly generated by Top1 at sites of genomic ribonucleotides. We show that RNase H2‐deficient yeast cells displayed elevated frequency of Rad52 foci, inactivation of RNase H2 and RAD52 led to synthetic lethality, and combined loss of RNase H2 and RAD51 induced slow growth and replication stress. Importantly, these phenotypes were rescued upon additional deletion of TOP1, implicating homologous recombination for the repair of Top1‐induced damage at ribonuclelotide sites. We demonstrate biochemically that irreversible DSBs are generated by subsequent Top1 cleavage on the opposite strand from the Top1‐induced DNA nicks at ribonucleotide sites. Analysis of Top1‐linked DNA from pull‐down experiments revealed that Top1 is covalently linked to the end of DNA in RNase H2‐deficient yeast cells, supporting this model. Taken together, these results define Top1 as a source of DSBs and genome instability when ribonucleotides incorporated by the replicative polymerases are not removed by RNase H2.     

Recognition of RNA by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid–specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.     

Ribodysgenesis: sudden genome instability in the yeast Saccharomyces cerevisiae arising from RNase H2 cleavage at genomic-embedded ribonucleotides

Ribonucleotides can be incorporated into DNA during replication by the replicative DNA polymerases. These aberrant DNA subunits are efficiently recognized and removed by Ribonucleotide Excision Repair, which is initiated by the heterotrimeric enzyme RNase H2. While RNase H2 is essential in higher eukaryotes, the yeast Saccharomyces cerevisiae can survive without RNase H2 enzyme, although the genome undergoes mutation, recombination and other genome instability events at an increased rate. Although RNase H2 can be considered as a protector of the genome from the deleterious events that can ensue from recognition and removal of embedded ribonucleotides, under conditions of high ribonucleotide incorporation and retention in the genome in a RNase H2-negative strain, sudden introduction of active RNase H2 causes massive DNA breaks and genome instability in a condition which we term ‘ribodysgenesis’. The DNA breaks and genome instability arise solely from RNase H2 cleavage directed to the ribonucleotide-containing genome. Survivors of ribodysgenesis have massive loss of heterozygosity events stemming from recombinogenic lesions on the ribonucleotide-containing DNA, with increases of over 1000X from wild-type. DNA breaks are produced over one to two divisions and subsequently cells adapt to RNase H2 and ribonucleotides in the genome and grow with normal levels of genome instability.     

RNase H is an exo- and endoribonuclease with asymmetric directionality,depending on the binding mode to the structural variants of RNA:DNA hybrids

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.     

RNA at DNA Double-Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

RNA polymerase II is recruited to DNA double-strand breaks (DSBs), transcribes the sequences that flank the break and produces a novel RNA type that has been termed damage-induced long non-coding RNA (dilncRNA). DilncRNAs can be processed into short, miRNA-like molecules or degraded by different ribonucleases. They can also form double-stranded RNAs or DNA:RNA hybrids. The DNA:RNA hybrids formed at DSBs contribute to the recruitment of repair factors during the early steps of homologous recombination (HR) and, in this way, contribute to the accuracy of the DNA repair. However, if not resolved, the DNA:RNA hybrids are highly mutagenic and prevent the recruitment of later HR factors. Here recent discoveries about the synthesis, processing, and degradation of dilncRNAs are revised. The focus is on RNA clearance, a necessary step for the successful repair of DSBs and the aim is to reconcile contradictory findings on the effects of dilncRNAs and DNA:RNA hybrids in HR.     

BRCA2 promotes DNA‐RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair

The BRCA2 tumor suppressor is a DNA double‐strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2‐deficient cells spontaneously accumulate DNA‐RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD‐box RNA helicase DDX5 as a BRCA2‐interacting protein. DDX5 associates with DNA‐RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA‐RNA hybrid‐unwinding activity of DDX5 helicase. An impaired BRCA2‐DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2‐T207A, reduces the association of DDX5 with DNA‐RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA‐RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.     

Loss of ribonuclease DIS3 hampers genome integrity in myeloma by disrupting DNA:RNA hybrid metabolism

The ribonuclease DIS3 is one of the most frequently mutated genes in the hematological cancer multiple myeloma, yet the basis of its tumor suppressor function in this disease remains unclear. Herein, exploiting the TCGA dataset, we found that DIS3 plays a prominent role in the DNA damage response. DIS3 inactivation causes genomic instability by increasing mutational load, and a pervasive accumulation of DNA:RNA hybrids that induces genomic DNA double‐strand breaks (DSBs). DNA:RNA hybrid accumulation also prevents binding of the homologous recombination (HR) machinery to double‐strand breaks, hampering DSB repair. DIS3‐inactivated cells become sensitive to PARP inhibitors, suggestive of a defect in homologous recombination repair. Accordingly, multiple myeloma patient cells mutated for DIS3 harbor an increased mutational burden and a pervasive overexpression of pro‐inflammatory interferon, correlating with the accumulation of DNA:RNA hybrids. We propose DIS3 loss in myeloma to be a driving force for tumorigenesis via DNA:RNA hybrid‐dependent enhanced genome instability and increased mutational rate. At the same time, DIS3 loss represents a liability that might be therapeutically exploited in patients whose cancer cells harbor DIS3 mutations.