Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

Chan, Yujia Alina; Aristizabal, Maria J.; Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michæl S.; Stirling, Peter C.; Hieter, Philip

doi:10.1371/journal.pgen.1004288

Cited by 203 publications

(262 citation statements)

References 78 publications

(113 reference statements)

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“…3). The observed pausing sites may be enriched at hot spots of Top1-DNA interaction sites and correspond to CPT-trapped Top1-DNA complexes, yet they correlated with potential sites of R-loop formation (37,38), such as the 3′ end of 35S genes (d in Fig. 3) or 5S genes (c in Fig.…”

Section: R-loops Promote Genome Instability and Noncanonical Replicationmentioning

confidence: 97%

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Stuckey

García‐Rodríguez

Aguilera

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.D uring transcription, DNA acts as a template for the synthesis of the nascent RNA. RNA synthesis is accompanied by the generation of positive and negative DNA supercoiling in front of and behind the transcription machinery, respectively (1). Unwinding of the DNA double helix by negative supercoiling may allow the RNA to hybridize to its DNA template behind the elongating RNA polymerase, leading to R-loops (2). Other elements that could potentiate R-loop accumulation include RNA: DNA hybrid-facilitating DNA sequences, such as G-quadruplex structures (3) or nicks in the nontemplate DNA strand (4).Eukaryotic cells need to control R-loop formation to avoid replication impairment, genome instability, and life span shortening mediated by such intermediates (5-10; reviewed in ref. 11). To do so, cells catalyze the relaxation of supercoiled DNA by type I topoisomerases (12-15), thus preventing replication fork reversal (16), DNA overwinding with the potential to block replication fork progression (17), DNA unwinding (18), or R-loop-mediated blocks of ribosomal RNA synthesis (19). Other enzymatic activities involved in R-loop processing include ribonuclease H (RNase H) activities, DNA-RNA helicases, such as Sen1/senataxin (20, 21), or Ataxin-2 RNA-binding protein Pbp1 (10). The ribonuclease activity of Saccharomyces cerevisiae RNases H1 and H2 specifically cleaves the RNA moiety of the RNA:DNA hybrid structure (22), whereas RNase H2 and topoisomerase 1 (Top1) can also process ribonucleotides in duplex DNA (23,24).Notably, R-loops are required to initiate mitochondrial DNA replication (25) and pioneering studies connected R-loops to origin-independent replication in prokaryotic systems (26,27). For example, DnaA-dependent initiation of DNA replication at the Escherichia coli oriC replication origin can be overcome in the absence of RNase H1 (28, 29). As a consequence, rnhA mutants can survive complete inactivation of oriC by transcriptiondependent activation of so-called oriK sites (30, 31), although candidate oriK sites have been identified only recently (32). Additional evidence for R-loop-primed replication was given by the observation that rnhA mutants are prone to an increase in mutation and DNA amplification events if origin activity is suppressed. These events required remov...

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Section: R-loops Promote Genome Instability and Noncanonical Replicationmentioning

confidence: 97%

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Stuckey

García‐Rodríguez

Aguilera

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…These mitotic recombination events presumably marked the sites of repair from damage induced by RNase H deficiency. No correlation was observed between the positions of these mitotic events and the positions of R-loop-prone regions that had been defined by a genome-wide tiling array map (8). This failure in correlation could be attributed both to the low number of events resulting from the genome-wide nature of study and to the low resolution of the R-loop map.…”

mentioning

confidence: 91%

Differential roles of the RNases H in preventing chromosome instability

Zimmer

Koshland

2016

Proc. Natl. Acad. Sci. U.S.A.

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DNA:RNA hybrids can lead to DNA damage and genome instability. This damage can be prevented by degradation of the RNA in the hybrid by two evolutionarily conserved enzymes, RNase H1 and H2. Indeed, RNase H-deficient cells have increased chromosomal rearrangements. However, the quantitative and spatial contributions of the individual enzymes to hybrid removal have been unclear. Additionally, RNase H2 can remove single ribonucleotides misincorporated into DNA during replication. The relative contribution of DNA:RNA hybrids and misincorporated ribonucleotides to chromosome instability also was uncertain. To address these issues, we studied the frequency and location of loss-ofheterozygosity (LOH) events on chromosome III in Saccharomyces cerevisiae strains that were defective for RNase H1, H2, or both. We showed that RNase H2 plays the major role in preventing chromosome III instability through its hybrid-removal activity. Furthermore, RNase H2 acts pervasively at many hybrids along the chromosome. In contrast, RNase H1 acts to prevent LOH within a small region of chromosome III where the instability is dependent upon two hybrid-prone sequences. This restriction of RNase H1 activity to a subset of hybrids is not the result of its constrained localization, because we found it at hybrids genome-wide. This result suggests that the genome-protection activity of RNase H1 is regulated at a step after hybrid recognition. The global function of RNase H2 and the region-specific function of RNase H1 provide insight into why these enzymes with overlapping hybrid-removal activities have been conserved throughout evolution.RNase H | R-loops | DNA:RNA hybrids | chromosome instability | genome instability P reventing chromosome instability is an essential process for maintaining genetic information. A source of chromosome instability is the accumulation of R-loops, which form when an RNA molecule hybridizes with a portion of genomic DNA, creating a DNA:RNA hybrid and a displaced single-stranded DNA (reviewed in ref. 1). One mechanism to prevent hybridmediated damage involves RNase H1 and RNase H2, two endogenous enzymes, conserved from bacteria to humans, that can degrade the RNA in R-loops (reviewed in ref.2). RNase H2 also functions in the removal of single ribonucleotides that are inappropriately incorporated into DNA by DNA polymerases during replication. Why RNase H1 and H2, which appear to have overlapping functions, remain highly conserved across many branches of life has been an outstanding question. Two areas of inquiry that will help address this conundrum are (i) does one of these RNases carry the major burden of preventing spontaneous R-loop-mediated chromosome instability, and, if so which, and (ii) do RNase H1 and H2 protect the same or different regions of the genome from R-loop-mediated damage?Whether RNase H1 and H2 contribute differentially to protecting against hybrid-mediated genome instability has been controversial. Studies of the inactivation of RNase H1 in yeast have shown little effect on chromosome insta...

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“…70,76,77 It is also likely that these R-loops are cleared by transcription of another lncRNA molecule. The latter is supported by the fact that high levels of transcription correlate with the presence of R-loops in wild type cells, 78 which would be predicted to interfere with transcription if these structures were static. If this is the case, one would predict that most chromatin-bound factors would be displaced by these R-loops, irrespective of their role in transcriptional regulation.…”

mentioning

confidence: 96%

LncRNAs: Bridging environmental sensing and gene expression

2016

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The survival of all organisms is dependent on complex, coordinated responses to environmental cues. Non-coding RNAs have been identified as major players in regulation of gene expression, with recent evidence supporting roles for long non-coding (lnc)RNAs in both transcriptional and post-transcriptional control. Evidence from our laboratory shows that lncRNAs have the ability to form hybridized structures called R-loops with specific DNA target sequences in S. cerevisiae, thereby modulating gene expression. In this Point of View, we provide an overview of the nature of lncRNA-mediated control of gene expression in the context of our studies using the GAL gene cluster as a model for controlling the timing of transcription.

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Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

Cited by 203 publications

References 78 publications

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Differential roles of the RNases H in preventing chromosome instability

LncRNAs: Bridging environmental sensing and gene expression

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