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

Replication Fork Reversal after Replication–Transcription Collision

Replication fork arrest is a recognized source of genetic instability, and transcription is one of the most prominent causes of replication impediment. We analyze here the requirement for recombination proteins in Escherichia coli when replication–transcription head-on collisions are induced at a specific site by the inversion of a highly expressed ribosomal operon (rrn). RecBC is the only recombination protein required for cell viability under these conditions of increased replication-transcription collisions. In its absence, fork breakage occurs at the site of collision, and the resulting linear DNA is not repaired and is slowly degraded by the RecJ exonuclease. Lethal fork breakage is also observed in cells that lack RecA and RecD, i.e. when both homologous recombination and the potent exonuclease V activity of the RecBCD complex are inactivated, with a slow degradation of the resulting linear DNA by the combined action of the RecBC helicase and the RecJ exonuclease. The sizes of the major linear fragments indicate that DNA degradation is slowed down by the encounter with another rrn operon. The amount of linear DNA decreases nearly two-fold when the Holliday junction resolvase RuvABC is inactivated in recB, as well as in recA recD mutants, indicating that part of the linear DNA is formed by resolution of a Holliday junction. Our results suggest that replication fork reversal occurs after replication–transcription head-on collision, and we propose that it promotes the action of the accessory replicative helicases that dislodge the obstacle

Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli

Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps

Branch Migration Prevents DNA Loss during Double-Strand Break Repair

The repair of DNA double-strand breaks must be accurate to avoid genomic rearrangements that can lead to cell death and disease. This can be accomplished by promoting homologous recombination between correctly aligned sister chromosomes. Here, using a unique system for generating a site-specific DNA double-strand break in one copy of two replicating Escherichia coli sister chromosomes, we analyse the intermediates of sister-sister double-strand break repair. Using two-dimensional agarose gel electrophoresis, we show that when double-strand breaks are formed in the absence of RuvAB, 4-way DNA (Holliday) junctions are accumulated in a RecG-dependent manner, arguing against the long-standing view that the redundancy of RuvAB and RecG is in the resolution of Holliday junctions. Using pulsed-field gel electrophoresis, we explain the redundancy by showing that branch migration catalysed by RuvAB and RecG is required for stabilising the intermediates of repair as, when branch migration cannot take place, repair is aborted and DNA is lost at the break locus. We demonstrate that in the repair of correctly aligned sister chromosomes, an unstable early intermediate is stabilised by branch migration. This reliance on branch migration may have evolved to help promote recombination between correctly aligned sister chromosomes to prevent genomic rearrangements

Avoiding chromosome pathology when replication forks collide

Chromosome duplication normally initiates via the assembly of replication fork complexes at defined origins(1,2). DNA synthesis by any one fork is thought to cease when it meets another travelling in the opposite direction, at which stage the replication machinery may simply dissociate before the nascent strands are finally ligated. But what actually happens is not clear. Here we present evidence consistent with the idea that every fork collision has the potential to threaten genomic integrity. In Escherichia coli this threat is kept at bay by RecG DNA translocase(3) and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet via a replisome assembly mechanism normally associated with fork repair, replication restart and recombination(4,5), establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realised when roadblocks to fork progression are reduced or eliminated. It relies on the chromosome being circular, reinforcing the idea that replication initiation is triggered repeatedly by fork collision. The results reported raise the question of whether replication fork collisions have pathogenic potential for organisms that exploit multiple origins to replicate each chromosome