Distribution of Holliday junctions and repair forks during Escherichia coli DNA double-strand break repair.

Accurate repair of DNA double-strand breaks (DSBs) is crucial for cell survival and genome integrity. In Escherichia coli, DSBs are repaired by homologous recombination (HR), using an undamaged sister chromosome as template. The DNA intermediates of this pathway are expected to be branched molecules that may include 4-way structures termed Holliday junctions (HJs), and 3-way structures such as D-loops and repair forks. Using a tool creating a site-specific, repairable DSB on only one of a pair of replicating sister chromosomes, we have determined how these branched DNA intermediates are distributed across a DNA region that is undergoing DSB repair. In cells, where branch migration and cleavage of HJs are limited by inactivation of the RuvABC complex, HJs and repair forks are principally accumulated within a distance of 12 kb from sites of recombination initiation, known as Chi, on each side of the engineered DSB. These branched DNA structures can even be detected in the region of DNA between the Chi sites flanking the DSB, a DNA segment not expected to be engaged in recombination initiation, and potentially degraded by RecBCD nuclease action. This is observed even in the absence of the branch migration and helicase activities of RuvAB, RadA, RecG, RecQ and PriA. The detection of full-length DNA fragments containing HJs in this central region implies that DSB repair can restore the two intact chromosomes, into which HJs can relocate prior to their resolution. The distribution of recombination intermediates across the 12kb region beyond Chi is altered in xonA, recJ and recQ mutants suggesting that, in the RecBCD pathway of DSB repair, exonuclease I stimulates the formation of repair forks and that RecJQ promotes strand-invasion at a distance from the recombination initiation sites.

Prevent and Cure: RPA Cooperates with Mre11-Sae2 in DNA Secondary Structure Repair.

DNA inversion duplications are genome rearrangements observed in cancer. In this issue, Deng et al. (2015) demonstrate that in S. cerevisiae RPA and Mre11-Sae2 cooperate to prevent the formation of inversion duplications initiated at short DNA secondary structures.

DNA double strand break repair in Escherichia coli perturbs cell division and chromosome dynamics.

To prevent the transmission of damaged genomic material between generations, cells require a system for accommodating DNA repair within their cell cycles. We have previously shown that Escherichia coli cells subject to a single, repairable site-specific DNA double-strand break (DSB) per DNA replication cycle reach a new average cell length, with a negligible effect on population growth rate. We show here that this new cell size distribution is caused by a DSB repair-dependent delay in completion of cell division. This delay occurs despite unperturbed cell size regulated initiation of both chromosomal DNA replication and cell division. Furthermore, despite DSB repair altering the profile of DNA replication across the genome, the time required to complete chromosomal duplication is invariant. The delay in completion of cell division is accompanied by a DSB repair-dependent delay in individualization of sister nucleoids. We suggest that DSB repair events create inter-sister connections that persist until those chromosomes are separated by a closing septum.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome.

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this "σ-replicating chromosome" causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.

RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification.

DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bacteria to humans rely on homologous recombination for accurate DSB repair. This requires both coordinate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA amplification and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Previous work has revealed recD mutants to be nuclease defective but recombination proficient. Despite this proficiency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromosome amplification and defective chromosome segregation. Our results demonstrate that RecBCD plays an important role in avoiding this amplification by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site.

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

RecG Directs DNA Synthesis during Double-Strand Break Repair.

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.

Chromosomal directionality of DNA mismatch repair in Escherichia coli.

Defects in DNA mismatch repair (MMR) result in elevated mutagenesis and in cancer predisposition. This disease burden arises because MMR is required to correct errors made in the copying of DNA. MMR is bidirectional at the level of DNA strand polarity as it operates equally well in the 5' to 3' and the 3' to 5' directions. However, the directionality of MMR with respect to the chromosome, which comprises parental DNA strands of opposite polarity, has been unknown. Here, we show that MMR in Escherichia coli is unidirectional with respect to the chromosome. Our data demonstrate that, following the recognition of a 3-bp insertion-deletion loop mismatch, the MMR machinery searches for the first hemimethylated GATC site located on its origin-distal side, toward the replication fork, and that resection then proceeds back toward the mismatch and away from the replication fork. This study provides support for a tight coupling between MMR and DNA replication.

Quantitative genomic analysis of RecA protein binding during DNA double-strand break repair reveals RecBCD action in vivo.

Understanding molecular mechanisms in the context of living cells requires the development of new methods of in vivo biochemical analysis to complement established in vitro biochemistry. A critically important molecular mechanism is genetic recombination, required for the beneficial reassortment of genetic information and for DNA double-strand break repair (DSBR). Central to recombination is the RecA (Rad51) protein that assembles into a spiral filament on DNA and mediates genetic exchange. Here we have developed a method that combines chromatin immunoprecipitation with next-generation sequencing (ChIP-Seq) and mathematical modeling to quantify RecA protein binding during the active repair of a single DSB in the chromosome of Escherichia coli. We have used quantitative genomic analysis to infer the key in vivo molecular parameters governing RecA loading by the helicase/nuclease RecBCD at recombination hot-spots, known as Chi. Our genomic analysis has also revealed that DSBR at the lacZ locus causes a second RecBCD-mediated DSBR event to occur in the terminus region of the chromosome, over 1 Mb away.

Long inverted repeat transiently stalls DNA replication by forming hairpin structures on both leading and lagging strands.

Long inverted repeats (LIRs), often found in eukaryotic genomes, are unstable in Escherichia coli where they are recognized by the SbcCD (the bacterial Mre11/Rad50 homologue), an endonuclease/exonuclease capable of cleaving hairpin DNA. It has long been postulated that LIRs form hairpin structures exclusively on the lagging-strand template during DNA replication, and SbcCD cleaves these hairpin-containing lagging strands to generate DNA double-strand breaks. Using a reconstituted oriC plasmid DNA replication system, we have examined how a replication fork behaves when it meets a LIR on DNA. We have shown that leading-strand synthesis stalls transiently within the upstream half of the LIR. Pausing of lagging-strand synthesis at the LIR was not clearly observed, but the pattern of priming sites for Okazaki fragment synthesis was altered within the downstream half of the LIR. We have found that the LIR on a replicating plasmid was cleaved by SbcCD with almost equal frequency on both the leading- and lagging-strand templates. These data strongly suggest that the LIR is readily converted to a cruciform DNA, before the arrival of the fork, creating SbcCD-sensitive hairpin structures on both leading and lagging strands. We propose a model for the replication-dependent extrusion of LIRs to form cruciform structures that transiently impede replication fork movement.

A novel mode of nuclease action is revealed by the bacterial Mre11/Rad50 complex.

The Mre11/Rad50 complex is a central player in various genome maintenance pathways. Here, we report a novel mode of nuclease action found for the Escherichia coli Mre11/Rad50 complex, SbcC2/D2 complex (SbcCD). SbcCD cuts off the top of a cruciform DNA by making incisions on both strands and continues cleaving the dsDNA stem at ∼10-bp intervals. Using linear-shaped DNA substrates, we observed that SbcCD cleaved dsDNA using this activity when the substrate was 110 bp long, but that on shorter substrates the cutting pattern was changed to that predicted for the activity of a 3'-5' exonuclease. Our results suggest that SbcCD processes hairpin and linear dsDNA ends with this novel DNA end-dependent binary endonuclease activity in response to substrate length rather than using previously reported activities. We propose a model for this mode of nuclease action, which provides new insight into SbcCD activity at a dsDNA end.

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.

SbcCD causes a double-strand break at a DNA palindrome in the Escherichia coli chromosome.

Long DNA palindromes are sites of genome instability (deletions, amplification, and translocations) in both prokaryotic and eukaryotic cells. In Escherichia coli, genetic evidence has suggested that they are sites of DNA cleavage by the SbcCD complex that can be repaired by homologous recombination. Here we obtain in vivo physical evidence of an SbcCD-induced DNA double-strand break (DSB) at a palindromic sequence in the E. coli chromosome and show that both ends of the break stimulate recombination. Cleavage is dependent on DNA replication, but the observation of two ends at the break argues that cleavage does not occur at the replication fork. Genetic analysis shows repair of the break requires the RecBCD recombination pathway and PriA, suggesting a mechanism of bacterial DNA DSB repair involving the establishment of replication forks.

A perfect palindrome in the Escherichia coli chromosome forms DNA hairpins on both leading- and lagging-strands.

DNA palindromes are hotspots for DNA double strand breaks, inverted duplications and intra-chromosomal translocations in a wide spectrum of organisms from bacteria to humans. These reactions are mediated by DNA secondary structures such as hairpins and cruciforms. In order to further investigate the pathways of formation and cleavage of these structures, we have compared the processing of a 460 base pair (bp) perfect palindrome in the Escherichia coli chromosome with the same construct interrupted by a 20 bp spacer to form a 480 bp interrupted palindrome. We show here that the perfect palindrome can form hairpin DNA structures on the templates of the leading- and lagging-strands in a replication-dependent reaction. In the presence of the hairpin endonuclease SbcCD, both copies of the replicated chromosome containing the perfect palindrome are cleaved, resulting in the formation of an unrepairable DNA double-strand break and cell death. This contrasts with the interrupted palindrome, which forms a hairpin on the lagging-strand template that is processed to form breaks, which can be repaired by homologous recombination.

WITHDRAWN: Symmetries and Asymmetries Associated with Non-Random Segregation of Sister DNA Strands in Escherichia coli.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.semcdb.2013.05.010. The duplicate article has therefore been withdrawn.

Symmetries and asymmetries associated with non-random segregation of sister DNA strands in Escherichia coli.

The successful inheritance of genetic information across generations is a complex process requiring replication of the genome and its faithful segregation into two daughter cells. At each replication cycle there is a risk that new DNA strands incorporate genetic changes caused by miscopying of parental information. By contrast the parental strands retain the original information. This raises the intriguing possibility that specific cell lineages might inherit "immortal" parental DNA strands via non-random segregation. If so, this requires an understanding of the mechanisms of non-random segregation. Here, we review several aspects of asymmetry in the very symmetrical cell, Escherichia coli, in the interest of exploring the potential basis for non-random segregation of leading- and lagging-strand replicated chromosome arms. These considerations lead us to propose a model for DNA replication that integrates chromosome segregation and genomic localisation with non-random strand segregation.

Non-random segregation of sister chromosomes in Escherichia coli.

It has long been known that the 5' to 3' polarity of DNA synthesis results in both a leading and lagging strand at all replication forks. Until now, however, there has been no evidence that leading or lagging strands are spatially organized in any way within a cell. Here we show that chromosome segregation in Escherichia coli is not random but is driven in a manner that results in the leading and lagging strands being addressed to particular cellular destinations. These destinations are consistent with the known patterns of chromosome segregation. Our work demonstrates a new level of organization relating to the replication and segregation of the E. coli chromosome.

DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat.

Approximately half the human genome is composed of repetitive DNA sequences classified into microsatellites, minisatellites, tandem repeats, and dispersed repeats. These repetitive sequences have coevolved within the genome but little is known about their potential interactions. Trinucleotide repeats (TNRs) are a subclass of microsatellites that are implicated in human disease. Expansion of CAG·CTG TNRs is responsible for Huntington disease, myotonic dystrophy, and a number of spinocerebellar ataxias. In yeast DNA double-strand break (DSB) formation has been proposed to be associated with instability and chromosome fragility at these sites and replication fork reversal (RFR) to be involved either in promoting or in preventing instability. However, the molecular basis for chromosome fragility of repetitive DNA remains poorly understood. Here we show that a CAG·CTG TNR array stimulates instability at a 275-bp tandem repeat located 6.3 kb away on the Escherichia coli chromosome. Remarkably, this stimulation is independent of both DNA double-strand break repair (DSBR) and RFR but is dependent on a functional mismatch repair (MMR) system. Our results provide a demonstration, in a simple model system, that MMR at one type of repetitive DNA has the potential to influence the stability of another. Furthermore, the mechanism of this stimulation places a limit on the universality of DSBR or RFR models of instability and chromosome fragility at CAG·CTG TNR sequences. Instead, our data suggest that explanations of chromosome fragility should encompass the possibility of chromosome gaps formed during MMR.

Novel roles for selected genes in meiotic DNA processing.

High-throughput studies of the 6,200 genes of Saccharomyces cerevisiae have provided valuable data resources. However, these resources require a return to experimental analysis to test predictions. An in-silico screen, mining existing interaction, expression, localization, and phenotype datasets was developed with the aim of selecting minimally characterized genes involved in meiotic DNA processing. Based on our selection procedure, 81 deletion mutants were constructed and tested for phenotypic abnormalities. Eleven (13.6%) genes were identified to have novel roles in meiotic DNA processes including DNA replication, recombination, and chromosome segregation. In particular, this analysis showed that Def1, a protein that facilitates ubiquitination of RNA polymerase II as a response to DNA damage, is required for efficient synapsis between homologues and normal levels of crossover recombination during meiosis. These characteristics are shared by a group of proteins required for Zip1 loading (ZMM proteins). Additionally, Soh1/Med31, a subunit of the RNA pol II mediator complex, Bre5, a ubiquitin protease cofactor and an uncharacterized protein, Rmr1/Ygl250w, are required for normal levels of gene conversion events during meiosis. We show how existing datasets may be used to define gene sets enriched for specific roles and how these can be evaluated by experimental analysis.

The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication.

It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.