Interplay between chromosomal architecture and termination of DNA replication in bacteria.

Faithful transmission of the genome from one generation to the next is key to life in all cellular organisms. In the majority of bacteria, the genome is comprised of a single circular chromosome that is normally replicated from a single origin, though additional genetic information may be encoded within much smaller extrachromosomal elements called plasmids. By contrast, the genome of a eukaryote is distributed across multiple linear chromosomes, each of which is replicated from multiple origins. The genomes of archaeal species are circular, but are predominantly replicated from multiple origins. In all three cases, replication is bidirectional and terminates when converging replication fork complexes merge and 'fuse' as replication of the chromosomal DNA is completed. While the mechanics of replication initiation are quite well understood, exactly what happens during termination is far from clear, although studies in bacterial and eukaryotic models over recent years have started to provide some insight. Bacterial models with a circular chromosome and a single bidirectional origin offer the distinct advantage that there is normally just one fusion event between two replication fork complexes as synthesis terminates. Moreover, whereas termination of replication appears to happen in many bacteria wherever forks happen to meet, termination in some bacterial species, including the well-studied bacteria Escherichia coli and Bacillus subtilis, is more restrictive and confined to a 'replication fork trap' region, making termination even more tractable. This region is defined by multiple genomic terminator (ter) sites, which, if bound by specific terminator proteins, form unidirectional fork barriers. In this review we discuss a range of experimental results highlighting how the fork fusion process can trigger significant pathologies that interfere with the successful conclusion of DNA replication, how these pathologies might be resolved in bacteria without a fork trap system and how the acquisition of a fork trap might have provided an alternative and cleaner solution, thus explaining why in bacterial species that have acquired a fork trap system, this system is remarkably well maintained. Finally, we consider how eukaryotic cells can cope with a much-increased number of termination events.

Cas1-Cas2 physically and functionally interacts with DnaK to modulate CRISPR Adaptation.

Prokaryotic Cas1-Cas2 protein complexes generate adaptive immunity to mobile genetic elements (MGEs), by capture and integration of MGE DNA in to CRISPR sites. De novo immunity relies on naive adaptation-Cas1-Cas2 targeting of MGE DNA without the aid of pre-existing immunity 'interference' complexes-by mechanisms that are not clear. Using E. coli we show that the chaperone DnaK inhibits DNA binding and integration by Cas1-Cas2, and inhibits naive adaptation in cells that results from chromosomal self-targeting. Inhibition of naive adaptation was reversed by deleting DnaK from cells, by mutation of the DnaK substrate binding domain, and by expression of an MGE (phage λ) protein. We also imaged fluorescently labelled Cas1 in living cells, observing that Cas1 foci depend on active DNA replication, and are much increased in frequency in cells lacking DnaK. We discuss a model in which DnaK provides a mechanism for restraining naive adaptation from DNA self-targeting, until DnaK is triggered to release Cas1-Cas2 to target MGE DNA.

Artificial sweeteners inhibit multidrug-resistant pathogen growth and potentiate antibiotic activity.

Antimicrobial resistance is one of the most pressing concerns of our time. The human diet is rich with compounds that alter bacterial gut communities and virulence-associated behaviours, suggesting food additives may be a niche for the discovery of novel anti-virulence compounds. Here, we identify three artificial sweeteners, saccharin, cyclamate and acesulfame-K (ace-K), that have a major growth inhibitory effect on priority pathogens. We further characterise the impact of ace-K on multidrug-resistant Acinetobacter baumannii, demonstrating that it can disable virulence behaviours such as biofilm formation, motility and the ability to acquire exogenous antibiotic-resistant genes. Further analysis revealed the mechanism of growth inhibition is through bulge-mediated cell lysis and that cells can be rescued by cation supplementation. Antibiotic sensitivity assays demonstrated that at sub-lethal concentrations, ace-K can resensitise A. baumannii to last resort antibiotics, including carbapenems. Using a novel ex vivo porcine skin wound model, we show that ace-K antimicrobial activity is maintained in the wound microenvironment. Our findings demonstrate the influence of artificial sweeteners on pathogen behaviour and uncover their therapeutic potential.

Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA.

The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the "replication fork trap" region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.

A Fork Trap in the Chromosomal Termination Area Is Highly Conserved across All Escherichia coli Phylogenetic Groups.

Termination of DNA replication, the final stage of genome duplication, is surprisingly complex, and failures to bring DNA synthesis to an accurate conclusion can impact genome stability and cell viability. In Escherichia coli, termination takes place in a specialised termination area opposite the origin. A 'replication fork trap' is formed by unidirectional fork barriers via the binding of Tus protein to genomic ter sites. Such a fork trap system is found in some bacterial species, but it appears not to be a general feature of bacterial chromosomes. The biochemical properties of fork trap systems have been extensively characterised, but little is known about their precise physiological roles. In this study, we compare locations and distributions of ter terminator sites in E. coli genomes across all phylogenetic groups, including Shigella. Our analysis shows that all ter sites are highly conserved in E. coli, with slightly more variability in the Shigella genomes. Our sequence analysis of ter sites and Tus proteins shows that the fork trap is likely to be active in all strains investigated. In addition, our analysis shows that the dif chromosome dimer resolution site is consistently located between the innermost ter sites, even if rearrangements have changed the location of the innermost termination area. Our data further support the idea that the replication fork trap has an important physiological role that provides an evolutionary advantage.

Too Much of a Good Thing: How Ectopic DNA Replication Affects Bacterial Replication Dynamics.

Each cell division requires the complete and accurate duplication of the entire genome. In bacteria, the duplication process of the often-circular chromosomes is initiated at a single origin per chromosome, resulting in two replication forks that traverse the chromosome in opposite directions. DNA synthesis is completed once the two forks fuse in a region diametrically opposite the origin. In some bacteria, such as Escherichia coli, the region where forks fuse forms a specialized termination area. Polar replication fork pause sites flanking this area can pause the progression of replication forks, thereby allowing forks to enter but not to leave. Transcription of all required genes has to take place simultaneously with genome duplication. As both of these genome trafficking processes share the same template, conflicts are unavoidable. In this review, we focus on recent attempts to add additional origins into various ectopic chromosomal locations of the E. coli chromosome. As ectopic origins disturb the native replichore arrangements, the problems resulting from such perturbations can give important insights into how genome trafficking processes are coordinated and the problems that arise if this coordination is disturbed. The data from these studies highlight that head-on replication-transcription conflicts are indeed highly problematic and multiple repair pathways are required to restart replication forks arrested at obstacles. In addition, the existing data also demonstrate that the replication fork trap in E. coli imposes significant constraints to genome duplication if ectopic origins are active. We describe the current models of how replication fork fusion events can cause serious problems for genome duplication, as well as models of how such problems might be alleviated both by a number of repair pathways as well as the replication fork trap system. Considering the problems associated both with head-on replication-transcription conflicts as well as head-on replication fork fusion events might provide clues of how these genome trafficking issues have contributed to shape the distinct architecture of bacterial chromosomes.

Direct removal of RNA polymerase barriers to replication by accessory replicative helicases.

Bacterial genome duplication and transcription require simultaneous access to the same DNA template. Conflicts between the replisome and transcription machinery can lead to interruption of DNA replication and loss of genome stability. Pausing, stalling and backtracking of transcribing RNA polymerases add to this problem and present barriers to replisomes. Accessory helicases promote fork movement through nucleoprotein barriers and exist in viruses, bacteria and eukaryotes. Here, we show that stalled Escherichia coli transcription elongation complexes block reconstituted replisomes. This physiologically relevant block can be alleviated by the accessory helicase Rep or UvrD, resulting in the formation of full-length replication products. Accessory helicase action during replication-transcription collisions therefore promotes continued replication without leaving gaps in the DNA. In contrast, DinG does not promote replisome movement through stalled transcription complexes in vitro. However, our data demonstrate that DinG operates indirectly in vivo to reduce conflicts between replication and transcription. These results suggest that Rep and UvrD helicases operate on DNA at the replication fork whereas DinG helicase acts via a different mechanism.

A role for 3' exonucleases at the final stages of chromosome duplication in Escherichia coli.

Chromosome duplication initiates via the assembly of replication fork complexes at defined origins, from where they proceed in opposite directions until they fuse with a converging fork. Recent work highlights that the completion of DNA replication is highly complex in both pro- and eukaryotic cells. In this study we have investigated how 3' and 5' exonucleases contribute towards the successful termination of chromosome duplication in Escherichia coli. We show that the absence of 3' exonucleases can trigger levels of over-replication in the termination area robust enough to allow successful chromosome duplication in the absence of oriC firing. Over-replication is completely abolished if replication fork complexes are prevented from fusing by chromosome linearization. Our data strongly support the idea that 3' flaps are generated as replication fork complexes fuse. In the absence of 3' exonucleases, such as ExoI, these 3' flaps can be converted into 5' flaps, which are degraded by 5' exonucleases, such as ExoVII and RecJ. Our data support the idea that multiple protein activities are required to process fork fusion intermediates. They highlight the complexity of fork fusions and further support the idea that the termination area evolved to contain fork fusion-mediated pathologies.

Origins Left, Right, and Centre: Increasing the Number of Initiation Sites in the Escherichia coli Chromosome.

The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication⁻transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY, integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replication⁻transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.

Replication-transcription conflicts trigger extensive DNA degradation in Escherichia coli cells lacking RecBCD.

Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD.

Chromosomal over-replication in Escherichia coli recG cells is triggered by replication fork fusion and amplified if replichore symmetry is disturbed.

Chromosome duplication initiates via the assembly of replication forks at defined origins. Forks proceed in opposite directions until they fuse with a converging fork. Recent work highlights that fork fusions are highly choreographed both in pro- and eukaryotic cells. The circular Escherichia coli chromosome is replicated from a single origin (oriC), and a single fork fusion takes place in a specialised termination area opposite oriC that establishes a fork trap mediated by Tus protein bound at ter sequences that allows forks to enter but not leave. Here we further define the molecular details of fork fusions and the role of RecG helicase in replication termination. Our data support the idea that fork fusions have the potential to trigger local re-replication of the already replicated DNA. In ΔrecG cells this potential is realised in a substantial fraction of cells and is dramatically elevated when one fork is trapped for some time before the converging fork arrives. They also support the idea that the termination area evolved to contain such over-replication and we propose that the stable arrest of replication forks at ter/Tus complexes is an important feature that limits the likelihood of problems arising as replication terminates.

Replication Termination: Containing Fork Fusion-Mediated Pathologies in Escherichia coli.

Duplication of bacterial chromosomes is initiated via the assembly of two replication forks at a single defined origin. Forks proceed bi-directionally until they fuse in a specialised termination area opposite the origin. This area is flanked by polar replication fork pause sites that allow forks to enter but not to leave. The precise function of this replication fork trap has remained enigmatic, as no obvious phenotypes have been associated with its inactivation. However, the fork trap becomes a serious problem to cells if the second fork is stalled at an impediment, as replication cannot be completed, suggesting that a significant evolutionary advantage for maintaining this chromosomal arrangement must exist. Recently, we demonstrated that head-on fusion of replication forks can trigger over-replication of the chromosome. This over-replication is normally prevented by a number of proteins including RecG helicase and 3' exonucleases. However, even in the absence of these proteins it can be safely contained within the replication fork trap, highlighting that multiple systems might be involved in coordinating replication fork fusions. Here, we discuss whether considering the problems associated with head-on replication fork fusion events helps us to better understand the important role of the replication fork trap in cellular metabolism.

25 years on and no end in sight: a perspective on the role of RecG protein.

The RecG protein of Escherichia coli is a double-stranded DNA translocase that unwinds a variety of branched substrates in vitro. Although initially associated with homologous recombination and DNA repair, studies of cells lacking RecG over the past 25 years have led to the suggestion that the protein might be multi-functional and associated with a number of additional cellular processes, including initiation of origin-independent DNA replication, the rescue of stalled or damaged replication forks, replication restart, stationary phase or stress-induced 'adaptive' mutations and most recently, naïve adaptation in CRISPR-Cas immunity. Here we discuss the possibility that many of the phenotypes of recG mutant cells that have led to this conclusion may stem from a single defect, namely the failure to prevent re-replication of the chromosome. We also present data indicating that this failure does indeed contribute substantially to the much-reduced recovery of recombinants in conjugational crosses with strains lacking both RecG and the RuvABC Holliday junction resolvase.

The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.

UNLABELLED: Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. IMPORTANCE: Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.

Shaping the landscape of the Escherichia coli chromosome: replication-transcription encounters in cells with an ectopic replication origin.

Each cell division requires the unwinding of millions of DNA base pairs to allow chromosome duplication and gene transcription. As DNA replication and transcription share the same template, conflicts between both processes are unavoidable and head-on collisions are thought to be particularly problematic. Surprisingly, a recent study reported unperturbed cell cycle progression in Escherichia coli cells with an ectopic replication origin in which highly transcribed rrn operons were forced to be replicated opposite to normal. In this study we have re-generated a similar strain and found the doubling time to be twice that of normal cells. Replication profiles of this background revealed significant deviations in comparison to wild-type profiles, particularly in highly transcribed regions and the termination area. These deviations were alleviated by mutations that either inactivate the termination area or destabilise RNA polymerase complexes and allow their easier displacement by replication forks. Our data demonstrate that head-on replication-transcription conflicts are highly problematic. Indeed, analysis of the replication profile of the previously published E. coli construct revealed a chromosomal rearrangement that alleviates replication-transcription conflicts in an intriguingly simple way. Our data support the idea that avoiding head-on collisions has significantly contributed to shaping the distinct architecture of bacterial chromosomes.

Cellular location and activity of Escherichia coli RecG proteins shed light on the function of its structurally unresolved C-terminus.

RecG is a DNA translocase encoded by most species of bacteria. The Escherichia coli protein targets branched DNA substrates and drives the unwinding and rewinding of DNA strands. Its ability to remodel replication forks and to genetically interact with PriA protein have led to the idea that it plays an important role in securing faithful genome duplication. Here we report that RecG co-localises with sites of DNA replication and identify conserved arginine and tryptophan residues near its C-terminus that are needed for this localisation. We establish that the extreme C-terminus, which is not resolved in the crystal structure, is vital for DNA unwinding but not for DNA binding. Substituting an alanine for a highly conserved tyrosine near the very end results in a substantial reduction in the ability to unwind replication fork and Holliday junction structures but has no effect on substrate affinity. Deleting or substituting the terminal alanine causes an even greater reduction in unwinding activity, which is somewhat surprising as this residue is not uniformly present in closely related RecG proteins. More significantly, the extreme C-terminal mutations have little effect on localisation. Mutations that do prevent localisation result in only a slight reduction in the capacity for DNA repair.

Avoiding chromosome pathology when replication forks collide.

Chromosome duplication normally initiates through the assembly of replication fork complexes at defined origins. 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 and by single-strand DNA exonucleases. Without RecG, replication initiates where forks meet through a replisome assembly mechanism normally associated with fork repair, replication restart and recombination, establishing new forks with the potential to sustain cell growth and division without an active origin. This potential is realized 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 several origins to replicate each chromosome.

On the viability of Escherichia coli cells lacking DNA topoisomerase I.

BACKGROUND: Manipulations of the DNA double helix during replication, transcription and other nucleic acid processing cause a change of DNA topology, which results in torsional stress. This stress is relaxed by DNA topoisomerases, a class of enzymes present in all domains of life. Negatively supercoiled DNA is relaxed by type IA topoisomerases that are widespread in bacteria, archaea and eukaryotes. In Escherichia coli there is conflicting data about viability of ΔtopA cells lacking topoisomerase I. RESULTS: In this study we sought to clarify whether E. coli cells lacking topoisomerase I are viable by using a plasmid-based lethality assay that allowed us to investigate the phenotype of ΔtopA cells without the presence of any compensatory mutations. Our results show that cells lacking topoisomerase I show an extreme growth defect and cannot be cultured without the accumulation of compensatory mutations. This growth defect can be partially suppressed by overexpression of topoisomerase III, the other type IA topoisomerase in E. coli, suggesting that the accumulation of torsional stress is, at least partially, responsible for the lethality of ΔtopA cells. The absence of RNase HI strongly exacerbates the phenotype of cells lacking topoisomerase I, which supports the idea that the processing of RNA:DNA hybrids is vitally important in ΔtopA cells. However, we did not observe suppression of the ΔtopA phenotype by increasing the level of R-loop processing enzymes, such as RNase HI or RecG. CONCLUSIONS: Our data show unambiguously that E. coli cells are not viable in the absence of DNA topoisomerase I without the presence of compensatory mutations. Furthermore, our data suggest that the accumulation of R-loops is not the primary reason for the severe growth defect of cells lacking topoisomerase I, which is in contrast to the current literature. Potential reasons for this discrepancy are discussed.

Localization of an accessory helicase at the replisome is critical in sustaining efficient genome duplication.

Genome duplication requires accessory helicases to displace proteins ahead of advancing replication forks. Escherichia coli contains three helicases, Rep, UvrD and DinG, that might promote replication of protein-bound DNA. One of these helicases, Rep, also interacts with the replicative helicase DnaB. We demonstrate that Rep is the only putative accessory helicase whose absence results in an increased chromosome duplication time. We show also that the interaction between Rep and DnaB is required for Rep to maintain rapid genome duplication. Furthermore, this Rep-DnaB interaction is critical in minimizing the need for both recombinational processing of blocked replication forks and replisome reassembly, indicating that colocalization of Rep and DnaB minimizes stalling and subsequent inactivation of replication forks. These data indicate that E. coli contains only one helicase that acts as an accessory motor at the fork in wild-type cells, that such an activity is critical for the maintenance of rapid genome duplication and that colocalization with the replisome is crucial for this function. Given that the only other characterized accessory motor, Saccharomyces cerevisiae Rrm3p, associates physically with the replisome, our demonstration of the functional importance of such an association indicates that colocalization may be a conserved feature of accessory replicative motors.

Budding yeast Mph1 promotes sister chromatid interactions by a mechanism involving strand invasion.

Stalling of replication forks at lesions is a serious threat to genomic integrity and cell viability. Cells have developed a variety of pathways that allow continuation of synthesis, including translesion synthesis, postreplication repair and homologous recombination. We have devised a sensitive genetic system for detection of sister chromatid interactions in Saccharomyces cerevisiae. A 266bp sequence duplication in the KanMX4 module was generated and reversions were scored via G418 resistant colonies. Both 4-NQO induced and spontaneous reversions are strictly dependent on RAD52. Damage-induced reversions are also largely dependent on RAD51. Thus, most damage-induced events require a strand invasion step. Induced reversions were not affected in rev3 mutants and partially reduced in rad30 mutants indicating an involvement of Pol η. In cells lacking Mph1, a member of the FANCM family of DNA helicases, that has been implicated in a pathway for fork reactivation involving homologous recombination, damage-induced events are significantly reduced. Together with the spontaneous mutator phenotype of mph1 mutants this data strongly suggest that Mph1 has an additional function in recombination besides its previously described ability to disrupt D-loops. We propose that Mph1 promotes D-loop formation.