YabA of Bacillus subtilis controls DnaA-mediated replication initiation but not the transcriptional response to replication stress

yabA encodes a negative regulator of replication initiation in Bacillus subtilis and homologues are found in many other Gram-positive species. YabA interacts with the β-processivity clamp (DnaN) of DNA polymerase and with the replication initiator and transcription factor DnaA. Because of these interactions, YabA has been proposed to modulate the activity of DnaA. We investigated the role of YabA in regulating replication initiation and the activity of DnaA as a transcription factor. We found that YabA function is mainly limited to replication initiation at oriC. Loss of YabA did not significantly alter expression of genes controlled by DnaA during exponential growth or after replication stress, indicating that YabA is not required for modulating DnaA transcriptional activity. We also found that DnaN activates replication initiation apparently through effects on YabA. Furthermore, association of GFP-YabA with the replisome correlated with the presence of DnaN at replication forks, but was independent of DnaA. Our results are consistent with models in which YabA inhibits replication initiation at oriC, and perhaps DnaA function at oriC, but not with models in which YabA generally modulates the activity of DnaA in response to replication stress.United States. Public Health Service (Grant GM41934)National Institutes of Health (U.S) ( Kirschstein NRSA postdoctoral fellowship 5 F32 G-076950

Co-directional replication-transcription conflicts lead to replication restart

August 24, 2011Head-on encounters between the replication and transcription machineries on the lagging DNA strand can lead to replication fork arrest and genomic instability1, 2. To avoid head-on encounters, most genes, especially essential and highly transcribed genes, are encoded on the leading strand such that transcription and replication are co-directional. Virtually all bacteria have the highly expressed ribosomal RNA genes co-directional with replication3. In bacteria, co-directional encounters seem inevitable because the rate of replication is about 10–20-fold greater than the rate of transcription. However, these encounters are generally thought to be benign2, 4, 5, 6, 7, 8, 9. Biochemical analyses indicate that head-on encounters10 are more deleterious than co-directional encounters8 and that in both situations, replication resumes without the need for any auxiliary restart proteins, at least in vitro. Here we show that in vivo, co-directional transcription can disrupt replication, leading to the involvement of replication restart proteins. We found that highly transcribed rRNA genes are hotspots for co-directional conflicts between replication and transcription in rapidly growing Bacillus subtilis cells. We observed a transcription-dependent increase in association of the replicative helicase and replication restart proteins where head-on and co-directional conflicts occur. Our results indicate that there are co-directional conflicts between replication and transcription in vivo. Furthermore, in contrast to the findings in vitro, the replication restart machinery is involved in vivo in resolving potentially deleterious encounters due to head-on and co-directional conflicts. These conflicts probably occur in many organisms and at many chromosomal locations and help to explain the presence of important auxiliary proteins involved in replication restart and in helping to clear a path along the DNA for the replisome.Biotechnology and Biological Sciences Research Council (Great Britain) (Grant BB/E006450/1)Wellcome Trust (London, England) (Grant 091968/Z/10/Z)National Institutes of Health (U.S.) (Grant GM41934)National Institutes of Health (U.S.) (Postdoctoral Fellowship GM093408)Biotechnology and Biological Sciences Research Council (Great Britain) (Sabbatical Visit

The <i>B</i>. <i>subtilis</i> Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units

<div><p>In bacteria the concurrence of DNA replication and transcription leads to potentially deleterious encounters between the two machineries, which can occur in either the head-on (lagging strand genes) or co-directional (leading strand genes) orientations. These conflicts lead to replication fork stalling and can destabilize the genome. Both eukaryotic and prokaryotic cells possess resolution factors that reduce the severity of these encounters. Though <i>Escherichia coli</i> accessory helicases have been implicated in the mitigation of head-on conflicts, direct evidence of these proteins mitigating co-directional conflicts is lacking. Furthermore, the endogenous chromosomal regions where these helicases act, and the mechanism of recruitment, have not been identified. We show that the essential <i>Bacillus subtilis</i> accessory helicase PcrA aids replication progression through protein coding genes of both head-on and co-directional orientations, as well as rRNA and tRNA genes. ChIP-Seq experiments show that co-directional conflicts at highly transcribed rRNA, tRNA, and head-on protein coding genes are major targets of PcrA activity on the chromosome. Partial depletion of PcrA renders cells extremely sensitive to head-on conflicts, linking the essential function of PcrA to conflict resolution. Furthermore, ablating PcrA’s ATPase/helicase activity simultaneously increases its association with conflict regions, while incapacitating its ability to mitigate conflicts, and leads to cell death. In contrast, disruption of PcrA’s C-terminal RNA polymerase interaction domain does not impact its ability to mitigate conflicts between replication and transcription, its association with conflict regions, or cell survival. Altogether, this work establishes PcrA as an essential factor involved in mitigating transcription-replication conflicts and identifies chromosomal regions where it routinely acts. As both conflicts and accessory helicases are found in all domains of life, these results are broadly relevant.</p></div

Replication–transcription conflicts in bacteria

DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separation of these two processes leads to their conflict, and failure to deal with this conflict can result in genome alterations and reduced fitness. In recent years major advances have been made in understanding how cells avoid conflicts between replication and transcription and how such conflicts are resolved when they do occur. In this Review, we summarize these findings, which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.National Institutes of Health (U.S.) (grant GM084003)National Institutes of Health (U.S.) (grant GM41934)National Institutes of Health (U.S.) (postdoctoral fellowship GM093408)University of Washington. Department of MicrobiologyCancer Prevention and Research Institute of Texas (Training Program grant RP101499

Strains used in this study.

<p>Strains used in this study.</p

PcrA is required for survival in the presence of severe head-on conflicts.

<p>A) Plating efficiency assays were carried out for strains containing the P<i>xis</i>-<i>lacZ</i> reporter constructs. 1:10 dilutions of exponential cultures were plated on agar plates with IPTG (at 2 μM or 100 μM, as indicated, leading to PcrA depletion) or without IPTG (no PcrA depletion). Transcription repression (Trx-, strain HM876) or de-repression (Trx+, strain HM877) due to the presence or absence, respectively, of the ImmR repressor protein is indicated below. Co-directional (CD) and head-on (HO) orientations of the reporters are indicated below the dilution series. B) Quantification of cell survival following PcrA depletion during <i>lacZ</i> expression (Trx+) is plotted. Percent survival of each strain containing the reporters, after IPTG-induced depletion of PcrA, relative to pre-depletion, was quantified and plotted (CD P<i>xis</i>-<i>lacZ</i> (gray) and HO P<i>xis</i>-<i>lacZ</i> (black)). Symbol * indicates that no colonies were detected after PcrA depletion with 100 μM IPTG in the presence of head-on <i>lacZ</i>. N = 6.</p

PcrA’s ATPase and helicase activity, but not its C-terminal domain, are required for conflict mitigation.

<p>A) Relative association of DnaC (compared to the control locus <i>yhaX</i>) was measured by ChIP-qPCRs. The endogenous co-directional (<i>rrn23S</i>, <i>rplGB</i>) and head-on (<i>dltB</i>) loci were analyzed without PcrA (empty vector control), in the presence of wild-type PcrA, a mutant of PcrA that should be incapable of interacting with RNA polymerase (PcrA-CΔ) or a PcrA allele lacking helicase and ATPase function (PcrA H-). B) Relative association of wild-type PcrA (compared to <i>yhaX</i>) or PcrA mutants (as well as empty vector control), as measured by ChIP-qPCRs, was determined for the same loci as in A. C) Plating efficiency assays were carried out for the PcrA degron strain expressing either an empty vector, wild-type PcrA (complementation of the PcrA degron strain), PcrA-CΔ, or PcrA H-. 10-fold dilutions of exponential cultures were plated either with (PcrA depletion at 100 μM, as indicated) or without (no PcrA depletion) IPTG. *P<0.05 and N>3.</p