Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

Lang, Kevin S.; Merrikh, Houra

doi:10.1101/691188

Cited by 9 publications

(10 citation statements)

References 73 publications

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“…Then, PcrA counteracts the accumulation of branched intermediates ( Figure 2 ) and disassembles RNA-DNA hybrids ( Figure 6 ), which accumulate in the Δ rnhC context as proposed in Figure 7 . RnhC, which interacts with a stalled RNAP [ 31 ], cleaves the RNA at the RNA-DNA hybrid substrate ( Figure 7 ) [ 84 ]. Then, PcrA bound to the ssDNA may dismantle the RNA-DNA hybrids to counteract the accumulation of branched intermediates in the absence of both PcrA and RnhC ( Figure 2 and Figure 7 ).…”

Section: Discussionmentioning

confidence: 99%

Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Álamo

Carrasco

Torres

et al. 2021

Cells

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Bacillus subtilis PcrA interacts with the RNA polymerase and might contribute to mitigate replication–transcription conflicts (RTCs). We show that PcrA depletion lethality is partially suppressed by rnhB inactivation, but cell viability is significantly reduced by rnhC or dinG inactivation. Following PcrA depletion, cells lacking RnhC or DinG are extremely sensitive to DNA damage. Chromosome segregation is not further impaired by rnhB or dinG inactivation but is blocked by rnhC or recA inactivation upon PcrA depletion. Despite our efforts, we could not construct a DrnhC DrecA strain. These observations support the idea that PcrA dismantles RTCs. Purified PcrA, which binds single-stranded (ss) DNA over RNA, is a ssDNA-dependent ATPase and preferentially unwinds DNA in a 3′®5′direction. PcrA unwinds a 3′-tailed RNA of an RNA-DNA hybrid significantly faster than that of a DNA substrate. Our results suggest that a replicative stress, caused by mis-incorporated rNMPs, indirectly increases cell viability upon PcrA depletion. We propose that PcrA, in concert with RnhC or DinG, contributes to removing spontaneous or enzyme-driven R-loops, to counteract deleterious trafficking conflicts and preserve to genomic integrity.

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Section: Discussionmentioning

confidence: 99%

Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Álamo

Carrasco

Torres

et al. 2021

Cells

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“…The transcription and replication machineries share the same DNA template, which renders head-on (HO) or codirectional (CD) collisions difficult to avoid 7,8 . As HO collisions are more deleterious than CD collisions, eukaryotic and prokaryotic cells have evolved strategies to prevent HO conflicts [9][10][11][12][13] , including a bias for the most transcribed genes towards a CD orientation with the direction of replication forks 8,[14][15][16] . However, the molecular consequences of frontal collisions between replication and transcription have remained largely unexplored at the genome-wide level in human cells.…”

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confidence: 99%

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

et al. 2020

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R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map 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 are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is 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 accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-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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“…Replication initiation from oriK45 would result in head-on collisions with RNA polymerases transcribing four rRNA operons carried between oriC and oriK45. Such head-on collisions are detrimental at least in part because of DNA topological issues that cause excessive R-loop formation in such conflict sites (42). The predominant suppressor found here would invert the DNA around oriC such that these four rRNA operons would now be on the leading strand of replication from oriK45.…”

Section: Discussionmentioning

confidence: 83%

Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

Veetil

Malhotra

Dubey

et al. 2020

mSphere

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The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

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Topological stress is responsible for the detrimental outcomes of head-on replication-transcription conflicts

Cited by 9 publications

References 73 publications

Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Bacillus subtilis PcrA Helicase Removes Trafficking Barriers

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

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