As the rates of DNA replication and transcription are different, the machineries that carry out these processes are bound to clash sometimes on the DNA helix. In contrast to results from head-on collisions, co-directional encounters have been shown to have mild effects in vitro, requiring no additional replication restart factors. Alan Grossman, Panos Soultanas and colleagues now show that in bacterial cells, highly transcribed genes are 'hotspots' for conflicts between transcription and replication. Such conflicts cause replication to pause or stall, and both types of events require the activities of restart proteins to resume replication. As the rates of replication and transcription are different, the machineries that carry out these processes are bound to clash on DNA. In contrast to results from head-on collisions, co-directional encounters have been shown to have mild effects in vitro, requiring no additional replication restart factors. It is now shown that in bacterial cells, both types of events require the activities of restart proteins to resume replication when a transcription complex is encountered. Head-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.

DNA footprinting and nuclease protection studies of PcrA helicase complexed with a 3′‐tailed DNA duplex reveal a contact region that covers a significant region of the substrate both in the presence and absence of a non‐hydrolysable analogue of ATP, ADPNP. However, details of the interactions of the enzyme with the duplex region are altered upon binding of nucleotide. By combining this information with that obtained from crystal structures of PcrA complexed with a similar DNA substrate, we have designed mutant proteins that are defective in helicase activity but that leave the ATPase and single‐stranded DNA translocation activities intact. These mutants are all located in domains 1B and 2B, which interact with the duplex portion of the DNA substrate. Taken together with the crystal structures, these data support an ‘active’ mechanism for PcrA that involves two distinct ATP‐dependent processes: destabilization of the duplex DNA ahead of the enzyme that is coupled to DNA translocation along the single strand product.

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.