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Abstract Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. Bdellovibrio bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living nonreplicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program. A predatory bacterium, Bdellovibrio bacteriovorus possesses a tightly packed chromosome and exhibits a unique “asynchronous mode” of replication (re)initiation.

The goal of this work is to propose a finite population counterpart to Eigen's model, which incorporates stochastic effects. The author considers a Moran model describing the evolution of a population of size m of chromosomes of length \\ell over an alphabet of cardinality \\kappa. The mutation probability per locus is q. He deals only with the sharp peak landscape: the replication rate is \\sigma>1 for the master sequence and 1 for the other sequences. He studies the equilibrium distribution of the process in the regime where \\ell\\to +\\infty,\\qquad m\\to +\\infty,\\qquad q\\to 0, {\\ell q} \\to a\\in ]0,+\\infty[, \\qquad\\frac{m}{\\ell}\\to\\alpha\\in [0,+\\infty].

Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis , the nucleoid occlusion protein Noc protects the chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP‐on‐Chip and bioinformatics, we have identified a consensus Noc‐binding DNA sequence (NBS), and have shown that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro . NBSs inserted near the replication terminus bound Noc–YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS array recruited Noc–YFP and conferred a severe Noc‐dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division, but that its activity is strictly localized by the interaction with NBS sites in vivo . We propose that Noc serves not only as a spatial regulator of cell division to protect the nucleoid, but also as a timing device with an important role in the coordination of chromosome segregation and cell division.

Propagation of genetic information is a fundamental prerequisite for living cells. We recently developed the replication cycle reaction (RCR), an in vitro reaction for circular DNA propagation, by reconstitution of the replication cycle of the Escherichia coli chromosome. In RCR, two replication forks proceed bidirectionally from the replication origin, oriC, and meet at a region opposite oriC, yielding two copies of circular DNA. Although RCR essentially propagates supercoiled monomers, concatemer byproducts are also produced due to inefficient termination of the replication fork progression. Here, we examined the effect of the Tus-ter replication fork trap in RCR. Unexpectedly, when the fork traps were placed opposite oriC, mimicking their arrangement on the chromosome, the propagation of circular DNA was inhibited. On the other hand, fork traps flanking oriC allowed efficient propagation of circular DNA and repressed concatemer production. These findings suggest that collision of the two convergence forks through the fork trap is detrimental to repetition of the replication cycle. We further demonstrate that this detrimental effect was rescued by the UvrD helicase. These results provide insights into the way in which circular DNA monomers replicate repetitively without generating concatemers.

A mechanism to explain chromosomal instability (CIN) in colorectal cancer is demonstrated; three new CIN-suppressor genes ( PIGN , MEX3C and ZNF516 ) encoded on chromosome 18q are identified, the loss of which leads to DNA replication stress, resulting in structural and numerical chromosome segregation errors, which are shown to be identical to phenotypes seen in CIN cells. Cause of chromosome instability in colorectal cancer Chromosomal instability (CIN) occurs in most solid tumours and is associated with poor prognosis and drug resistance. This study demonstrates a link between CIN in colorectal cancer and the loss of a region on chromosome 18q. The authors identify three previously unknown CIN-suppressor genes in this region that, when lost, lead to replication stress resulting in structural and numerical chromosome segregation errors. Supplementing tumour cell lines with nucleosides alleviates replication-associated damage, limits chromosome segregation errors after CIN-suppressor gene silencing and attenuates segregation errors and DNA damage in CIN + cells. These findings point to a genetic mechanism — distinct from mitotic defects — that causes chromosome instability in colorectal tumours and that might be pharmacologically reversible. Cancer chromosomal instability (CIN) results in an increased rate of change of chromosome number and structure and generates intratumour heterogeneity 1 , 2 . CIN is observed in most solid tumours and is associated with both poor prognosis and drug resistance 3 , 4 . Understanding a mechanistic basis for CIN is therefore paramount. Here we find evidence for impaired replication fork progression and increased DNA replication stress in CIN + colorectal cancer (CRC) cells relative to CIN − CRC cells, with structural chromosome abnormalities precipitating chromosome missegregation in mitosis. We identify three new CIN-suppressor genes ( PIGN (also known as MCD4 ), MEX3C ( RKHD2 ) and ZNF516 ( KIAA0222 )) encoded on chromosome 18q that are subject to frequent copy number loss in CIN + CRC. Chromosome 18q loss was temporally associated with aneuploidy onset at the adenoma–carcinoma transition. CIN-suppressor gene silencing leads to DNA replication stress, structural chromosome abnormalities and chromosome missegregation. Supplementing cells with nucleosides, to alleviate replication-associated damage 5 , reduces the frequency of chromosome segregation errors after CIN-suppressor gene silencing, and attenuates segregation errors and DNA damage in CIN + cells. These data implicate a central role for replication stress in the generation of structural and numerical CIN, which may inform new therapeutic approaches to limit intratumour heterogeneity.

Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA.

The 3D organization of mammalian chromatin was described more than 30 years ago by visualizing sites of DNA synthesis at different times during the S phase of the cell cycle. These early cytogenetic studies revealed structurally stable chromosome domains organized into subnuclear compartments. Active-gene-rich domains in the nuclear interior replicate early, whereas more condensed chromatin domains that are largely at the nuclear and nucleolar periphery replicate later. During the past decade, this spatiotemporal DNA replication programme has been mapped along the genome and found to correlate with epigenetic marks, transcriptional activity and features of 3D genome architecture such as chromosome compartments and topologically associated domains. But the causal relationship between these features and DNA replication timing and the regulatory mechanisms involved have remained an enigma. The recent identification of cis-acting elements regulating the replication time and 3D architecture of individual replication domains and of long non-coding RNAs that coordinate whole chromosome replication provide insights into such mechanisms.

Key Points Chromothripsis is a phenomenon by which tens to thousands of chromosomal rearrangements occur, with the available evidence indicating that chromothripsis can be generated by a single catastrophic event during the life history of a cell. Rearrangements can occur by chromosome shattering and rejoining of pieces by end-joining DNA repair pathways, or by aberrant DNA replication-based mechanisms. Chromothripsis may contribute to cellular transformation, as it occurs early in tumour development: end-joining-based repair can lead to the loss of tumour suppressor functions, oncogenic fusions and oncogene amplification via double-minute chromosomes. In addition, aberrant DNA replication mechanisms taking place during chromothripsis can lead to oncogene amplification. An attractive model for the generation of chromothripsis invokes the involvement of micronuclei. According to this model, chromosomes contained within micronuclei suffer aberrant DNA replication and can then be pulverized in mitosis with subsequent rejoining of DNA segments leading to a derivative chromosome or chromosomes that can be reincorporated into the main nucleus. Chromothripsis is observed with a higher frequency in cells with mutated p53. This leads to a model in which micronuclei formation owing to chromosome segregation errors is allowed in p53-deficient cells, potentially yielding chromothripsis and the evolution of cancer. Defects in chromosome segregation and/or DNA damage response processes may also contribute to carcinogenesis by promoting chromothripsis. Chromothripsis is an emerging phenomenon that results in chromosome rearrangements in tumour cells. This Review discusses the possible mechanisms underlying this process and its implications for cancer biology and in the clinic. Genomic alterations that lead to oncogene activation and tumour suppressor loss are important driving forces for cancer development. Although these changes can accumulate progressively during cancer evolution, recent studies have revealed that many cancer cells harbour chromosomes bearing tens to hundreds of clustered genome rearrangements. In this Review, we describe how this striking phenomenon, termed chromothripsis, is likely to arise through chromosome breakage and inaccurate reassembly. We also discuss the potential diagnostic, prognostic and therapeutic implications of chromothripsis in cancer.

A study of DNA replication timing in mouse and human cells reveals that replication domains (domains of the genome which replicate at the same time) share a correlation with topologically associating domains; these results reconcile cell-type-specific sub-nuclear compartmentalization with developmentally stable chromosome domains and offer a unified model for large scale chromosome structure and function. A unified model for large-scale chromosome organization As part of the mouse ENCODE project, David Gilbert and colleagues study the relationship between replication timing and higher order chromatin domains in mouse and human. They find that boundaries of replication domains — domains within the genome which replicate at the same time — share a near one-to-one correlation with topology associated domains. These and other results reconcile cell-type specific sub-nuclear compartmentalization with developmentally stable chromosome domains and offer a unified model for large-scale chromosome structure and function. Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program 1 . In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400–800 kilobases (‘replication domains’), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements 2 , 3 , 4 , 5 , 6 , 7 . Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs) 4 , 5 , 8 , 9 . Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains 8 , 10 . However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale 11 , 12 . Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure 8 , 9 , 13 . Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of [almost equal to] 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting [almost equal to] 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.