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DNA sequencing coverage in wild type and RNase H deficient Bacillus subtilis cells [1]

ABSTRACT: RNA-DNA hybrids form throughout the chromosome during normal cell growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, increase mutagenesis, and reduce gene expression. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion of RNA-DNA hybrids. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore resulting in SOS-dependent inhibition of cell division. We conclude that B. subtilis NCIB 3610 encodes functional RNase HI, HII, and HIII, and pBS32-encoded RNase HI contributes to replication fork progression and chromosome stability while RNase HIII is important for chromosome stability and plasmid hyper-replication.

ORGANISM(S): Bacillus subtilis subsp. subtilis NCIB 3610 = ATCC 6051 = DSM 10

PROVIDER: GSE149552 | GEO | 2020/04/30

REPOSITORIES: GEO

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DNA sequencing coverage in wild type and RNase H deficient Bacillus subtilis cells [2]

Project description:RNA-DNA hybrids form throughout the chromosome during normal cell growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, increase mutagenesis, and reduce gene expression. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion of RNA-DNA hybrids. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore resulting in SOS-dependent inhibition of cell division. We conclude that B. subtilis NCIB 3610 encodes functional RNase HI, HII, and HIII, and pBS32-encoded RNase HI contributes to replication fork progression and chromosome stability while RNase HIII is important for chromosome stability and plasmid hyper-replication.

2020-07-16 | GSE154585 | GEO

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Project description:R-loops represent a major source of replication stress but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest and restart in S. cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate normally their chromosomes under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Indeed, these cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative step in resuming replication. Similar impairments in nascent DNA resection at HU-arrested forks were observed in human cells lacking RNase H2. However, the addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation, fully restored fork resection. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

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Project description:We mapped the location of RNA:DNA hybrids in HeLa cells that had been knocked down with a control siRNA. We found that the levels of RNA:DNA hybrids around origins of replication (defined from data from Pretyk et al, Nat Commun 2016) strongly correlated to the directionality of transcription around these origins (defined from GRO-seq data from GEO dataset GSM1518913.) Specifically, in regions where the replication fork travels antiparallel (Head-On) to the direction of transcription we find elevated RNA:DNA hybrids, and in regions where the replication fork travels parallel to the direction of replication (Co-directional) we find reduced RNA:DNA hybrids. These results imply that the relative directions of replication and transcription have an effect on the levels of RNA:DNA hybrids across the genome.

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Project description:R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, we have mapped RNA:DNA hybrids, replication stress markers and DNA double strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids were found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR was only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also cumulated at TTS, leading to persistent checkpoint activation, spreading of -H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

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Senataxin associates with replication forks to protect fork integrity across RNAPII transcribed genes

Project description:Here we analysed the role of yeast Senataxin (Sen1) in coordinating replication with transcription and in protecting genome integrity. Senataxin is mutated in the two severe neurodegenerative diseases AOA2 and ALS4. We show that a fraction of Sen1/Senataxin DNA/RNA helicase associates with replication forks and protects the integrity of those fork encountering highly expressed RNAPII genes. sen1 mutants accumulate aberrant DNA structures and RNA-DNA hybrids while forks clash head-on with RNAPII transcription units and counteract recombinogenic events and accumulation of checkpoint signals. Nrd1, which acts togheter with Sen1 in trascription temination, is not recruited at replication forks. nrd1 mutants does not display replication defects, high genome instability and checkpoint activation observed in sen1 mutants The Sen1 function in replication can be therefore separable from its role in RNA processing. We propose a role for Sen1/Senataxin during chromosome replication in facilitating replisome progression across RNAPII transcribed genes thus preventing DNA-RNA hybrids accumulation when forks encounter nascent transcripts on the lagging strand template.

2012-11-09 | GSE39139 | GEO

Topoisomerase 1 dependent R-loop deficiency drives accelerated replication and genomic instability

Project description:DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression leading to aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results reveal a novel mechanism of oncogene-induced DNA damage induced by aberrant replication fork acceleration.

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Senataxin associates with replication forks to protect fork integrity across RNAPII transcribed genes

2012-11-09 | E-GEOD-39139 | biostudies-arrayexpress

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Project description:R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co- transcriptionally in the opposite orientation to replication fork progression. Recent studies have shown that replication forks stalled by co-transcription R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds RNA:DNA hybrids in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4 pathway for resolution of R-loop-mediated transcription- replication conflicts, which may be involved in R-loop unwinding.

2022-11-02 | PXD034331 | Pride

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2013-07-22 | E-GEOD-41975 | biostudies-arrayexpress

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