Chromosome segregation

The ability to visualise specific genes and proteins within bacterial cells is revolutionising knowledge of chromosome segregation. The essential elements appear to be the driving force behind DNA replication, which occurs at fixed cellular positions, the condensation of newly replicated DNA by a chromosome condensation machine located at the cell 1/4 and 3/4 positions, and molecular machines that act at midcell to allow chromosome separation after replication and movement of the sister chromosomes away from the division septum prior to cell division. This review attempts to provide a perspective on current views of the bacterial chromosome segregation mechanism and how it relates to other cellular processes.     

The Escherichia coli SMC Complex, MukBEF, Shapes Nucleoid Organization Independently of DNA Replication

SMC (structural maintenance of chromosomes) complexes function ubiquitously in organizing and maintaining chromosomes. Functional fluorescent derivatives of the Escherichia coli SMC complex, MukBEF, form foci that associate with the replication origin region (ori). MukBEF impairment results in mispositioning of ori and other loci in steady-state cells. These observations led to an earlier proposal that MukBEF positions new replicated sister oris. We show here that MukBEF generates and maintains the cellular positioning of chromosome loci independently of DNA replication. Rapid impairment of MukBEF function by depleting a Muk component in the absence of DNA replication leads to loss of MukBEF foci as well as mispositioning of ori and other loci, while rapid Muk synthesis leads to rapid MukBEF focus formation but slow restoration of normal chromosomal locus positioning.     

Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories

Faithful DNA replication ensures genetic integrity in eukaryotic cells, but it is still obscure how replication is organized in space and time within the nucleus. Using timelapse microscopy, we have developed a new assay to analyze the dynamics of DNA replication both spatially and temporally in individual Saccharomyces cerevisiae cells. This allowed us to visualize replication factories, nuclear foci consisting of replication proteins where the bulk of DNA synthesis occurs. We show that the formation of replication factories is a consequence of DNA replication itself. Our analyses of replication at specific DNA sequences support a long-standing hypothesis that sister replication forks generated from the same origin stay associated with each other within a replication factory while the entire replicon is replicated. This assay system allows replication to be studied at extremely high temporal resolution in individual cells, thereby opening a window into how replication dynamics vary from cell to cell.     

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.     

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed.     

Extent of the activity domain and possible roles of FtsK in the Escherichia coli chromosome terminus

Escherichia coli FtsK protein couples cell division and chromosome segregation. It is a component of the septum essential for cell division. It also acts during chromosome dimer resolution by XerCD-specific recombination at the dif site, with two distinct activities: DNA translocation oriented by skewed sequence elements and direct activation of Xer recombination. Dimer resolution requires that the skewed elements polarize in opposite directions 30-50 kb on either side of dif. This constitutes the DIF domain, approximately coincident with the region where replication terminates. The observation that the ftsK1 mutation increases recombination near dif was exploited to determine whether the chromosome region on which FtsK acts is limited to the DIF domain. A monitoring of recombination activity at multiple loci in a 350 kb region to the left of dif revealed (i) zones of differing activities unconnected to dimer resolution and (ii) a constant 10-fold increase of recombination in the 250 kb region adjacent to dif in the ftsK1 mutant. The latter effect allows definition of an FTSK domain whose total size is at least fourfold that of the DIF domain. Additional analyses revealed that FtsK activity responds to polarization in the whole FTSK domain and that displacement of the region where replication terminates preserves differences between recombination zones. Our interpretation is that translocation by FtsK occurs mostly on DNA belonging to a specifically organized domain of the chromosome, when physical links between either dimeric or still intercatenated chromosomes force this DNA to run across the septum at division.     

Independent positioning and action of Escherichia coli replisomes in live cells

A prevalent view of DNA replication has been that it is carried out in fixed "replication factories." By tracking the progression of sister replication forks with respect to genetic loci in live Escherichia coli, we show that at initiation replisomes assemble at replication origins irrespective of where the origins are positioned within the cell. Sister replisomes separate and move to opposite cell halves shortly after initiation, migrating outwards as replication proceeds and both returning to midcell as replication termination approaches. DNA polymerase is maintained at stalled replication forks, and over short intervals of time replisomes are more dynamic than genetic loci. The data are inconsistent with models in which replisomes associated with sister forks act within a fixed replication factory. We conclude that independent replication forks follow the path of the compacted chromosomal DNA, with no structure other than DNA anchoring the replisome to any particular cellular region.     

Structure and Function of RecQ DNA Helicases

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.     

Structure and function of RecQ DNA helicases

RecQ family helicases play important roles in coordinating genome maintenance pathways in living cells. In the absence of functional RecQ proteins, cells exhibit a variety of phenotypes, including increased mitotic recombination, elevated chromosome missegregation, hypersensitivity to DNA-damaging agents, and defects in meiosis. Mutations in three of the five human RecQ family members give rise to genetic disorders associated with a predisposition to cancer and premature aging, highlighting the importance of RecQ proteins and their cellular activities for human health. Current evidence suggests that RecQ proteins act at multiple steps in DNA replication, including stabilization of replication forks and removal of DNA recombination intermediates, in order to maintain genome integrity. The cellular basis of RecQ helicase function may be explained through interactions with multiple components of the DNA replication and recombination machinery. This review focuses on biochemical and structural aspects of the RecQ helicases and how these features relate to their known cellular function, specifically in preventing excessive recombination.     

Delineation of DNA replication time zones by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to visualize specific genomic DNA sequences in interphase nuclei. In normal diploid cells, unreplicated DNA segments give singlet hybridization signals while replicated loci are characterized by doublets. The distribution of these two patterns in unsynchronized cell populations can be used to determine the S phase replication time of any DNA sequence. The validity of this approach was established by analyzing genes whose replication profiles in expressing and non-expressing cells had been determined previously by conventional methods. Using this technique it has been possible to map the replication timing topography of the DNA within and flanking the cystic fibrosis (CF) gene locus on chromosome 7. The gene itself is located within a defined time zone which is approximately 500 kb in length and is under developmental control. It is early replicating in cells which express CF but late replicating in other cell types. These time zones probably represent basic units of chromosome structure.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

Protein interaction networks in bacteria

The complement of expressed cellular proteins - the proteome - is organized into functional, structured networks of protein interactions that mediate assembly of molecular machines and dynamic cellular pathways. Recent studies reveal the biological roles of protein interactions in bacteriophage T7 and Helicobacter pylori, and new methods allow to compare and to predict interaction networks in other species. Smaller scale networks provide biological insights into DNA replication and chromosome dynamics in Bacillus subtilis and Archeoglobus fulgidus, and into the assembly of multiprotein complexes such as the type IV secretion system of Agrobacterium tumefaciens, and the cell division machinery of Escherichia coli. Genome-wide interaction networks in several species are needed to obtain a biologically meaningful view of the higher order organization of the proteome in bacteria.     

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.     

Spatiotemporal dynamics of regulatory protein recruitment at DNA damage sites

Mammalian cells are constantly threatened by multiple types of DNA lesions arising from various sources like irradiation, environmental agents, replication errors or by-products of the normal cellular metabolism. If not readily detected and repaired these lesions can lead to cell death or to the transformation of cells giving rise to life-threatening diseases like cancer. Multiple specialized repair pathways have evolved to preserve the genetic integrity of a cell. The increasing number of DNA damage sensors, checkpoint regulators, and repair factors identified in the numerous interconnected repair pathways raises the question of how DNA repair is coordinated. In the last decade, various methods have been developed that allow the induction of DNA lesions and subsequent real-time analysis of repair factor assembly at DNA repair sites in living cells. This combination of biophysical and molecular cell biology methods has yielded interesting new insights into the order and kinetics of protein recruitment and identified regulatory sequences and selective loading platforms for the efficient restoration of the genetic and epigenetic integrity of mammalian cells.     

Epstein-Barr virus BGLF4 kinase induces premature chromosome condensation through activation of condensin and topoisomerase II

Previous studies of Epstein-Barr virus (EBV) replication focused mainly on the viral and cellular factors involved in replication compartment assembly and controlling the cell cycle. However, little is known about how EBV reorganizes nuclear architecture and the chromatin territories. In EBV-positive nasopharyngeal carcinoma NA cells or Akata cells, we noticed that cellular chromatin becomes highly condensed upon EBV reactivation. In searching for the possible mechanisms involved, we found that transient expression of EBV BGLF4 kinase induces unscheduled chromosome condensation, nuclear lamina disassembly, and stress fiber rearrangements, independently of cellular DNA replication and Cdc2 activity. BGLF4 interacts with condensin complexes, the major components in mitotic chromosome assembly, and induces condensin phosphorylation at Cdc2 consensus motifs. BGLF4 also stimulates the decatenation activity of topoisomerase II, suggesting that it may induce chromosome condensation through condensin and topoisomerase II activation. The ability to induce chromosome condensation is conserved in another gammaherpesvirus kinase, murine herpesvirus 68 ORF36. Together, these findings suggest a novel mechanism by which gammaherpesvirus kinases may induce multiple premature mitotic events to provide more extrachromosomal space for viral DNA replication and successful egress of nucleocapsid from the nucleus.     

Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli

To demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated. After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome. In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay. These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated. Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus. This supports the translocating replication apparatuses model.     

DNA motifs that sculpt the bacterial chromosome

During the bacterial cell cycle, the processes of chromosome replication, DNA segregation, DNA repair and cell division are coordinated by precisely defined events. Tremendous progress has been made in recent years in identifying the mechanisms that underlie these processes. A striking feature common to these processes is that non-coding DNA motifs play a central part, thus 'sculpting' the bacterial chromosome. Here, we review the roles of these motifs in the mechanisms that ensure faithful transmission of genetic information to daughter cells. We show how their chromosomal distribution is crucial for their function and how it can be analysed quantitatively. Finally, the potential roles of these motifs in bacterial chromosome evolution are discussed.