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

A conditional depletion system for PcrA.

<p>A) Fusion protein PcrA-ssrA is conditionally depleted following transcriptional induction of the adaptor protein gene <i>sspB</i>, via 100 μM IPTG treatment. A representative western blot probed for native PcrA shows approximately 90% depletion following IPTG addition (PcrA- vs. PcrA+) versus the non-specific band near the bottom of the gel which was used as a loading control. The black arrow indicates the location of PcrA on the blot. PcrA levels in a wild-type strain are shown in lane 2 (WT), and are equivalent to levels in the degron strain prior to depletion (PcrA+). Specificity of the polyclonal anti-PcrA antibody is demonstrated by the lack of signal in a <i>pcrA</i> deletion strain, which is suppressed by <i>recF</i> deletion. B) PcrA depletion (on plates containing 100 μM IPTG) is lethal, and is rescued by <i>recF</i> deletion. Top row: Negative control strain harboring P<i>spank</i>-<i>sspB</i> only. Middle row: PcrA Degron strain harboring both P<i>spank</i>-<i>sspB</i> and <i>pcrA-ssrA</i>. Lower row: PcrA degron and Δ<i>recF</i>.</p

Models for centromere-mediated early origin activation.

<p>(A) Kinetochore/microtubule interaction orients the centromere and pericentric DNA near the microtubule organizing center (MTOC) where there is an enrichment of replication initiation factors. (B) Tension exerted by the kinetochore/microtubule interaction induces an altered chromatin structure of pericentric DNA that provides accessibility of embedded origins to initiation factors. (C) Kinetochore proteins interact directly (or indirectly) with origin initiation factors recruiting them to nearby origins. (D) The organization of pericentric DNA into the C-loop orients origins within the C-loop to the periphery of the chromatin mass increasing their accessibility to initiation factors.</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

PcrA reduces DnaC association with engineered conflict regions.

<p>A) The P<i>xis</i>-<i>lacZ</i> reporter (gray arrow) and MLS resistance gene (black triangle) were integrated onto the chromosome either co-directionally (CD), or head-on (HO) to replication at the <i>thrC</i> locus (upstream and downstream <i>thrC</i> fragments used for integration into the chromosome are shown in white). P<i>spank(hy)</i>-<i>hisC</i> constructs share the same conceptual design, but have a spectinomycin resistance gene in place of the MLS gene and are integrated at <i>amyE</i>. B) mRNA levels were determined by RT-qPCR using primers that bind in the middle of <i>hisC</i> or <i>lacZ</i>. Levels are shown relative to the control gene, <i>yhaX</i>. “Trx” refers to transcription before or after induction with IPTG (Trx- and Trx+, respectively). C) The relative association of DnaC with either CD or HO <i>hisC</i> was determined by ChIP-qPCR and plotted relative to its association with control region <i>yhaX</i>. D) The relative association of DnaC with <i>lacZ</i> was determined as in 2C. Here <i>lacZ</i> is expressed/repressed in strains lacking/possessing repressor protein ImmR, respectively. “R” refers to inhibition of transcription by subsequent addition of rifampicin). “Rep.” refers to unperturbed or HPUra-inhibited replication. An additional condition is shown where DnaC association with <i>lacZ</i> was determined after replication was inhibited by 15 minutes of HPUra treatment (last bar on the right in the <i>lacZ</i> “HO” panel).</p

Replication dynamics for chromosome XIV in WT and rearranged strains.

<p>(A) Replication kinetic profiles of chromosome XIV in WT (top) and rearranged (bottom) strains. Percent replication was monitored across chromosome XIV at 40 (magenta), 45 (orange), 55 (green), and 65 (blue) minutes following release from alpha factor arrest. When the native centromere (yellow circle) is present near ARS1426, a prominent peak is seen in the 40 and 45 minute time samples. In this strain, the peak at ARS1410 is shallow in the 40 and 45 minute samples. When the centromere is repositioned (orange circle) near ARS1410 in the rearranged strain, both the time of appearance and the prominence of the peaks at ARS1410 and ARS1426 are inverted with respect to the WT strain. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s006" target="_blank">Figures S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s007" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s001" target="_blank">Datasets S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s002" target="_blank">S2</a> for all chromosomes. (B) Z-score plots of chromosome XIV in WT (black) and rearranged (blue) strains. Replication kinetic profiles from the 40 minute sample were normalized by converting percent replication values to Z-score values (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#s4" target="_blank">Materials and Methods</a>). Genomic loci corresponding to ARS1410 and ARS1426 show significant differences in Z-scores. ARS1424 is the next closest active origin to the endogenous centromere residing ∼19 kb to the left. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s008" target="_blank">Figures S4</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s009" target="_blank">S5</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s010" target="_blank">S6</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s003" target="_blank">Datasets S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s004" target="_blank">S4</a> for all chromosomes and the 45- and 65-minute samples.</p

Z-score and 2D gel analysis of ARS1531.

<p>(A) Z-score plot of chromosome XV in WT (black) and rearranged (blue) strains. ARS1531 displayed a difference of Z-score values at least as large as that seen for ARS1410 and ARS1426 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen-1002677-g005" target="_blank">Figure 5B</a>). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002677#pgen.1002677.s008" target="_blank">Figure S4</a> for all chromosomes. (B) 2D gel analysis of ARS1531 in the WT (left), the rearranged strain used in microarray analysis (middle), and the rearranged strain used for prior slot blot analysis (right). DNA from all three strains was digested with NcoI and BglII to give a 3.18 kb fragment harboring ARS1531 and then subjected to 2D gel analysis. The presence of a bubble arc in the WT (black arrow) indicates that ARS1531 is a functional origin in this strain. The presence of a bubble arc in one of the two rearranged strains confirms that the absence of origin activity in rearranged A (used in microarray analysis) is not due to relocation of the centromere on chromosome XIV. Below each 2D gel image is the sequence for the WT or mutant (red) ACS.</p

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast

<div><p>A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (<i>ORC1</i>, <i>ORC4</i>, <i>ORC6</i>, <i>CDC6</i>, <i>CDT1</i>, and <i>MCM5</i>). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and <i>CDC45</i>). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human <i>ORC4</i> alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (<i>orc4</i>^Y232C). We find that yeast cells with the <i>orc4</i>^Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, <i>orc4</i>^Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis.</p></div