The B. subtilis Accessory Helicase PcrA Facilitates DNA Replication through Transcription Units

Abstract

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 Escherichia coli 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 Bacillus subtilis 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.

Fig 1. A conditional depletion system for PcrA.

A) Fusion protein PcrA-ssrA is conditionally depleted following transcriptional induction of the adaptor protein gene sspB, 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 pcrA deletion strain, which is suppressed by recF deletion. B) PcrA depletion (on plates containing 100 μM IPTG) is lethal, and is rescued by recF deletion. Top row: Negative control strain harboring Pspank-sspB only. Middle row: PcrA Degron strain harboring both Pspank-sspB and pcrA-ssrA. Lower row: PcrA degron and ΔrecF.

Fig 2. PcrA reduces DnaC association with engineered conflict regions.

A) The Pxis-lacZ 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 thrC locus (upstream and downstream thrC fragments used for integration into the chromosome are shown in white). Pspank(hy)-hisC constructs share the same conceptual design, but have a spectinomycin resistance gene in place of the MLS gene and are integrated at amyE. B) mRNA levels were determined by RT-qPCR using primers that bind in the middle of hisC or lacZ. Levels are shown relative to the control gene, yhaX. “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 hisC was determined by ChIP-qPCR and plotted relative to its association with control region yhaX. D) The relative association of DnaC with lacZ was determined as in 2C. Here lacZ 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 lacZ was determined after replication was inhibited by 15 minutes of HPUra treatment (last bar on the right in the lacZ “HO” panel).

Fig 3. Replication intermediates accumulate at conflict sites.

A) Genomic DNA from PcrA degron strains HM876 (Trx-), or HM877 (Trx+) was enzymatically digested for 2D gel analysis: two discrete fragments were produced, both of which encoded the 3’ end of the lacZ gene which was targeted for qPCR analysis in Fig 2. The location (in base pairs) of the lacZ 3’ end within each fragment is indicated. B) 2D gel analysis of the AscI/ClaI or EagI/ApaLI digested fragment containing the lacZ 3`end and surrounding region. The approximate location of the lacZ 3`end is indicated with a white arrow, and the relative 1N spot intensity is indicated at the lower left (useful as a loading control). An incomplete digestion product (black spot) is present along the arc of linears in the EagI/ApaLI digest. C) Quantification of signal intensity along the Y-arc of the AscI/ClaI digestion fragment. D) Quantificaiton of the Y-arc of the EagI/ApaLI fragment. A magnified view of the region of the arc corresponding to the 3`end of lacZ is shown at the right.

Fig 4. PcrA associates with engineered conflict regions.

A) PcrA association was determined by ChIP of PcrA followed by qPCR for hisC in the presence (+IPTG) or absence (-IPTG) of transcription (Trx+, or Trx-, respectively) and following transcriptional induction and subsequent inhibition by rifampicin (Trx- “R”). B) ChIP-PCR of PcrA was performed as in 4A, but for lacZ. *P<0.05 and **P<0.01.

Fig 5. PcrA associates with, and reduces DnaC association with, endogenous conflict regions.

A) ChIP-Seq data showing chromosomal locations where DnaC association increases after PcrA depletion (strain HM448). Data were calculated by first normalizing ChIP-Seq samples to inputs. Normalized ChIP signal when PcrA was present was then subtracted from ChIP signal after PcrA depletion. The resulting differential signal is shown. Peaks at rRNA genes and tRNA genes are indicated. Selected peaks are labelled according to orientation. C = co-directional, H = head-on: ribosomal protein genes at 8–13° position (C1), ssbA (C2), pit (H1), cotC (H2), yoaM (H3), rpsD (H4), thrC (H5), amtB (H6), dltA-E (H7). The ter region is indicated by *. B) ChIP-Seq of Myc-PcrA in an otherwise wild-type strain containing the myc-pcrA allele (HM224). ChIP data were normalized to input, then non-specific peaks (identified via antibody control ChIP-Seq) were subtracted out. Peaks at rRNA and tRNA genes are indicated.

Fig 6. PcrA and DnaC association with specific chromosomal regions are transcription-dependent and correlate with RpoC association.

A) Relative association of PcrA with nine candidate loci, compared to the control locus yhaX was measured by ChIP-qPCR, with active (white bars, before rifampicin treatment) and inhibited (gray bars, after 3 min. of rifampicin treatment) transcription. B) Relative association of DnaC (compared to yhaX) as measured by ChIP-qPCR in the presence of PcrA and transcription (no rifampicin, black), following PcrA depletion (no rifampicin, white), and following PcrA depletion and transcription shut off (3 min. of rifampicin treatment, gray bars). C) Relative association of RpoC-GFP with the candidate regions (compared to yhaX) was measured by ChIP-qPCR before (white) and after transcription inhibition with rifampicin (gray). Co-directional and head-on genes are indicated above the graph. Pearson correlation coefficient for DnaC (-PcrA) vs. PcrA association = 0.7709. Pearson correlation coefficient for co-directional genes: R = 0.7 for DnaC (-PcrA) vs. RpoC, and R = 0.9 for PcrA vs. RpoC. N ≥ 5. *P<0.05 and **P<0.01.

Fig 7. PcrA is required for survival in the presence of severe head-on conflicts.

A) Plating efficiency assays were carried out for strains containing the Pxis-lacZ 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 lacZ 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 Pxis-lacZ (gray) and HO Pxis-lacZ (black)). Symbol * indicates that no colonies were detected after PcrA depletion with 100 μM IPTG in the presence of head-on lacZ. N = 6.

Fig 8. PcrA’s ATPase and helicase activity, but not its C-terminal domain, are required for conflict mitigation.

A) Relative association of DnaC (compared to the control locus yhaX) was measured by ChIP-qPCRs. The endogenous co-directional (rrn23S, rplGB) and head-on (dltB) 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 yhaX) 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.