Co-directional replication-transcription conflicts lead to replication restart

Abstract

Head-on encounters between the replication and transcription machineries on the lagging DNA strand can lead to replication fork arrest and genomic instability. 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 replication. 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 benign. Biochemical analyses indicate that head-on encounters are more deleterious than co-directional encounters 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.

Fig. 1

Head-on conflicts between transcription and replication cause increased association of helicase loader protein DnaB. Association of DnaB was assessed in ChIP-chip experiments in strains containing Pxis-lacZ inserted at thrC. Cells were grown in rich medium (LB) and sampled during exponential growth. The relative enrichment of a given chromosomal position is plotted on the y-axis vs the chromosomal position on the x-axis (in bp clockwise from oriC). Data are shown for the chromosomal region from ~3,240 kb through ~3,400 kb. The location of Pxis-lacZ inserted at thrC is indicated. Pxis-lacZ is head-on with replication. a. Data from cells expressing Pxis-lacZ (strain JMA264). b. Data from cells not expressing Pxis-lacZ (strain JMA201). These findings were verified by qPCR (Supplementary Fig. 3).

Fig. 2

ChIP-chip analysis of DnaB. Wild type cells (strain 168) were grown in LB (a) or defined minimal medium (b) and sampled during exponential growth. Data are plotted as in Fig. 1, except the chromosomal positions are shown from 0 kb (oriC) to just past rrnI, H, G at ~200 kb. Similar results were obtained at each identical rrn with both DnaD and DnaB, indicating the reproducibility of the data. Results were also confirmed by qPCR with independent samples from different strains (Fig. 3). Data from other rrn regions are presented in Supplementary Information (Supplementary Fig. 5). The rrn sequences represent a consensus and were thus presented as identical. For clarity and simplicity, we unambiguously label each individual locus according to its chromosomal location.

Fig. 3

Association of helicase loader proteins DnaD and DnaB with rrn loci depends on transcription and the replication restart protein priA. Wild type cells (AG174) and the priA-ssrA* mutant (WKS338) were grown to mid-exponential phase in LB medium. For wild type, samples were taken in the absence (black bars) or 4 min after treatment (gray bars) with rifampicin (30 μg/ml) to block transcription initiation. The priA-ssrA* mutant (grown in the presence of 1 μg/ml of chloramphenicol to maintain selection for the mutant allele) was sampled in the absence of rifampicin (white bars). Association of DnaD (a) and DnaB (b) was analysed by ChIP-qPCR with three different primer pairs (Supplementary Fig. 2) that recognize the indicated rrn loci. The ChIP-qPCR signals are normalized to gene copy number (Methods), so that the signal for the 23S rrn probe, which should detect all 10 rrn loci, is normalized per locus. Data are averages from at least three independent cultures. Error bars represent standard error.

Fig. 4

Association of the replicative helicase with rrn loci depends on transcription and is independent of dnaA. Samples from wild type cells (AG174) with and without rifampicin (Rif) and a dnaA null mutant (AIG200) were grown and analysed as described for Fig. 3. Data are averages from at least three independent cultures. Error bars represent standard error. We also tested association of DnaB with the rrn loci using the 23S rrn probe and found similar association in the dnaA null mutant (data not shown).