RecA finds homologous DNA by reduced dimensionality search

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3′ single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms.

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.

Genomic contacts reveal the control of sister chromosome decatenation in E. coli

In bacteria, chromosome segregation occurs progressively, from the origin to the terminus, a few minutes after the replication of each locus. In-between replication and segregation, sister loci are maintained in an apparent cohesive state by topological links. Whereas topoisomerase IV (Topo IV), the main bacteria decatenase, controls segregation, little is known regarding the influence of the cohesion step on chromosome folding. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, a massive chromosome reorganization takes place, associated with increases in trans-contacts between catenated sister chromatids and in long-range cis-contacts between the terminus and distant loci on the genome. A genetic analysis of these signals allowed us to decipher specific roles for Topo IV and Topo III, an accessory decatenase. Moreover we revealed the role of MatP, the terminus macrodomain organizing system and MukB, the E. coli SMC in organizing sister chromatids tied by persistent catenation links . We propose that large-scale conformation changes observed in these conditions reveal a defective decatenation hub located in the terminus area. Altogether, our findings support a model of spatial and temporal partition of the tasks required for sister chromosome segregation.

Kinetochore-independent mechanisms of sister chromosome separation

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.

Cohesion of Sister Chromosome Termini during the Early Stages of Sporulation in Bacillus subtilis

ABSTRACT During sporulation of Bacillus subtilis, the cell cycle is reorganized to generate separated prespore and mother cell compartments, each containing a single fully replicated chromosome. The process begins with reorganization of the nucleoid to form an elongated structure, the axial filament, in which the two chromosome origins are attached to opposite cell poles, with the remainder of the DNA stretched between these sites. When the cell then divides asymmetrically, the division septum closes around the chromosome destined for the smaller prespore, trapping the origin-proximal third of the chromosome in the prespore. A translocation pore is assembled through which a DNA transporter, SpoIIIE/FtsK, transfers the bulk of the chromosome to complete the segregation process. Although the mechanisms involved in attaching origin regions to the cell poles are quite well understood, little is known about other aspects of axial filament morphology. We have studied the behavior of the terminus region of the chromosome during sporulation using time-lapse imaging of wild-type and mutant cells. The results suggest that the elongated structure involves cohesion of the terminus regions of the sister chromosomes and that this cohesion is resolved when the termini reach the asymmetric septum or translocation pore. Possible mechanisms and roles of cohesion and resolution are discussed. IMPORTANCE Endospore formation in Firmicutes bacteria provides one of the most highly resistant life forms on earth. During the early stages of endospore formation, the cell cycle is reorganized so that exactly two fully replicated chromosomes are generated, before the cell divides asymmetrically to generate the prespore and mother cell compartments that are critical for the developmental process. Decades ago, it was discovered that just prior to asymmetrical division the two chromosomes enter an unusual elongated configuration called the axial filament. This paper provides new insights into the nature of the axial filament structure and suggests that cohesion of the normally separated sister chromosome termini plays an important role in axial filament formation.

Kinetochore-independent mechanisms of sister chromosome separation

ABSTRACTAlthough kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore-independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.AUTHOR SUMMARYKinetochores, the site on the chromosomes to which microtubules attach driving the separation and segregation of replicated sister chromosomes, have been viewed as essential for proper cell division and accurate transmission of chromosomes into daughter cells. However previous studies demonstrated that sister chromosomes lacking kinetochores (acentrics) often undergo separation, segregation and transmission. Here we demonstrate that sister acentrics are held together through DNA intertwining. We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers. We found that acentric sister chromatids display unique patterns of separation using mechanisms independent of the kinetochore. Additionally, we identified the specific microtubule-associated proteins required for the successful mitotic transmission of acentric chromosomes to daughter cells. These studies reveal unsuspected, distinct forces that likely act on all chromosomes during mitosis independent of kinetochore-microtubule attachments.

Evolutionary repair: changes in multiple functional modules allow meiotic cohesin to support mitosis

Different members of the same protein family often perform distinct cellular functions. How much are these differing functions due to changes in a protein’s biochemical activity versus changes in other proteins? We asked how the budding yeast, Saccharomyces cerevisiae, evolves when forced to use the meiosis-specific kleisin, Rec8, instead of the mitotic kleisin, Scc1, during the mitotic cell cycle. This perturbation impairs sister chromosome linkage and reduces reproductive fitness by 45%. We evolved 15 populations for 1750 generations, substantially increasing their fitness, and analyzed their genotypes and phenotypes. We found no mutations in Rec8, but many populations had mutations in the transcriptional mediator complex, cohesin-related genes, and cell cycle regulators that induce S phase. These mutations improve sister chromosome cohesion and slow genome replication in Rec8-expressing cells. We conclude that changes in known and novel partners allow proteins to improve their ability to perform new functions.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35–38 Amino Acids of GyrA

Prokaryotes have an essential gene—gyrase—that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35–38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to β-pinwheel elements is presented.

Dynamics of RecA-mediated repair of replication-dependent DNA breaks

Chromosomal replication is the major source of spontaneous DNA double-strand breaks (DSBs) in living cells. Repair of these DSBs is essential for cell viability, and accuracy of repair is critical to avoid chromosomal rearrangements. Repair of replication-dependent DSBs occurs primarily by homologous recombination with a sister chromosome. However, this reaction has never been visualized at a defined chromosomal locus, so little is known about its spatial or temporal dynamics. Repair of a replication-independent DSB generated in Escherichia coli by a rare-cutting endonuclease leads to the formation of a bundle of RecA filaments. In this study, we show that in contrast, repair of a replication-dependent DSB involves a transient RecA focus localized in the central region of the cell in which the DNA is replicated. The recombining loci remain centrally located with restricted movement before segregating with little extension to the period of postreplicative sister-chromosome cohesion. The spatial and temporal efficiency of this reaction is remarkable.