RESUMO
Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA+ ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability.
Assuntos
Mitose , Proteínas , Proteínas/genética , Adenosina Trifosfatases/metabolismo , DNA , Cromátides/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Replicação do DNA/genética , Dano ao DNARESUMO
Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS.
Assuntos
Replicação do DNA , Mitose , DNARESUMO
Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution.
RESUMO
Polo-like kinase 1 (PLK1) plays a fundamental role in the spatiotemporal control of mitosis. Cells lacking PLK1 activity exhibit characteristic chromosome misalignment due to defects in microtubule-kinetochore organization and attachment. In our recently published paper, we uncover a new role for PLK1 in the preservation and maintenance of centromere integrity.
RESUMO
Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment. Here, we reveal a previously undescribed mechanism named 'centromere disintegration' that drives chromosome misalignment in PLK1-inactivated cells. We find that PLK1 inhibition does not necessarily compromise metaphase establishment, but instead its maintenance. We demonstrate that this is caused by unlawful unwinding of DNA by BLM helicase at a specific centromere domain underneath kinetochores. Under bipolar spindle pulling, the distorted centromeres are promptly decompacted into DNA threadlike molecules, leading to centromere rupture and whole-chromosome arm splitting. Consequently, chromosome alignment collapses. Our study unveils an unexpected role of PLK1 as a chromosome guardian to maintain centromere integrity for chromosome biorientation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/fisiologia , Linhagem Celular , Pareamento Cromossômico/fisiologia , Humanos , Cinetocoros , Interferência de RNA , Timidina/farmacologia , Quinase 1 Polo-LikeRESUMO
Accurate duplication and transmission of identical genetic information into offspring cells lies at the heart of a cell division cycle. During the last stage of cellular division, namely mitosis, the fully replicated DNA molecules are condensed into X-shaped chromosomes, followed by a chromosome separation process called sister chromatid disjunction. This process allows for the equal partition of genetic material into two newly born daughter cells. However, emerging evidence has shown that faithful chromosome segregation is challenged by the presence of persistent DNA intertwining structures generated during DNA replication and repair, which manifest as so-called ultra-fine DNA bridges (UFBs) during anaphase. Undoubtedly, failure to disentangle DNA linkages poses a severe threat to mitosis and genome integrity. This review will summarize the possible causes of DNA bridges, particularly sister DNA inter-linkage structures, in an attempt to explain how they may be processed and how they influence faithful chromosome segregation and the maintenance of genome stability.
RESUMO
As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin's recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin's hinge driven by cycles of ATP hydrolysis.
Assuntos
Proteínas de Ciclo Celular/química , Cromátides/química , Proteínas Cromossômicas não Histona/química , DNA/química , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Cromatina/química , Humanos , Hidrólise , Lisina/química , Camundongos , Mutação , Proteínas Nucleares/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging. In contrast to conventional anaphase bridge-breakage models, we demonstrate that chromatid axes of the intertwined sister-chromatids rupture prior to the breakage of the DNA bridges. Consequently, the ruptured sister arms remain tethered and cause signature chromosome rearrangements, including whole-arm (Robertsonian-like) translocation/deletion and isochromosome formation. Therefore, our study reveals a hitherto unreported chromatid damage phenomenon mediated by sister DNA intertwinements that may help to explain the development of complex karyotypes in tumour cells.
Assuntos
Cromátides/genética , Quebras de DNA , Troca de Cromátide Irmã/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Instabilidade Cromossômica , Aberrações Cromossômicas , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/patologia , Fase S/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin's Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin's association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase-independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilação , Sítios de Ligação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA Fúngico/metabolismo , Modelos Moleculares , Mutação , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio - OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this 'internal standard' calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics.
Assuntos
Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/análise , Genoma Fúngico , Calibragem , Candida glabrata/genética , Ciclo Celular , Proteínas de Ciclo Celular/análise , Imunoprecipitação da Cromatina/normas , Proteínas Cromossômicas não Histona/análise , Proteínas Fúngicas/análise , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Mutantes/análise , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , CoesinasRESUMO
Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation. We show here that the N-terminal domain of yeast Scc1 contains two α helices, forming a four-helix bundle with the coiled coil emerging from Smc3's adenosine triphosphatase head. Mutations affecting this interaction compromise cohesin's association with chromosomes. The interface is far from Smc3 residues, whose acetylation prevents cohesin's dissociation from chromosomes. Cohesin complexes holding chromatids together in vivo do indeed have the configuration of hetero-trimeric rings, and sister DNAs are entrapped within these.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/química , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Reagentes de Ligações Cruzadas/química , Cristalografia por Raios X , DNA/química , Mutação , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Sister chromatid cohesion involves entrapment of sister DNAs by a cohesin ring created through association of a kleisin subunit (Scc1) with ATPase heads of Smc1/Smc3 heterodimers. Cohesin's association with chromatin involves subunits recruited by Scc1: Wapl, Pds5, and Scc3/SA, in addition to Scc2/4 loading complex. Unlike Pds5, Wapl, and Scc2/4, Scc3s are encoded by all eukaryotic genomes. Here, a crystal structure of Scc3 reveals a hook-shaped protein composed of tandem α helices. Its N-terminal domain contains a conserved and essential surface (CES) present even in organisms lacking Pds5, Wapl, and Scc2/4, while its C-terminal domain binds a section of the kleisin Scc1. Scc3 turns over in G2/M while maintaining cohesin's association with chromosomes and it promotes de-acetylation of Smc3 upon Scc1 cleavage.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química , Acetilação , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Cromossômicas não Histona/química , Pontos de Checagem da Fase G2 do Ciclo Celular , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteólise , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined. Here, we report that the DNA structure-specific nuclease MUS81-EME1 localizes to CFS loci in early mitotic cells, and promotes the cytological appearance of characteristic gaps or breaks observed at CFSs in metaphase chromosomes. These data indicate that CFS breakage is an active, MUS81-EME1-dependent process, and not a result of inadvertent chromatid rupturing during chromosome condensation. Moreover, CFS cleavage by MUS81-EME1 promotes faithful sister chromatid disjunction. Our findings challenge the prevailing view that CFS breakage is a nonspecific process that is detrimental to cells, and indicate that CFS cleavage actually promotes genome stability.
Assuntos
Sítios Frágeis do Cromossomo/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Regulação da Expressão Gênica , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Segregação de Cromossomos , Endodesoxirribonucleases/metabolismo , Imunofluorescência , Instabilidade Genômica , Humanos , Reação em Cadeia da PolimeraseRESUMO
Cohesin's Smc1 and Smc3 subunits form V-shaped heterodimers, the nucleotide binding domains (NBDs) of which bind the C- and N-terminal domains, respectively, of the α-kleisin subunit, forming a large tripartite ring within in which sister DNAs are entrapped, and thereby held together (sister chromatid cohesion). During replication, establishment of stable cohesion is dependent on Eco1-mediated acetylation of Smc3's NBD, which is thought to prevent dissociation of α-kleisin from Smc3, thereby locking shut a "DNA exit gate." How Scc3 and Pds5, regulatory subunits bound to α-kleisin, regulate cohesion establishment and maintenance is poorly understood. We show here that by binding to α-kleisin adjacent to its Smc3 nucleotide binding N-terminal domain, Pds5 not only promotes cohesin's release from chromatin but also mediates de novo acetylation of Smc3 by Eco1 during S phase and subsequently prevents de-acetylation by the deacetylase Hos1/HDAC8. By first promoting cohesin's release from chromosomes and subsequently creating and guarding the chemical modification responsible for blocking release, Pds5 enables chromosomal cohesin to switch during S phase from a state of high turnover to one capable of tenaciously holding sister chromatids together for extended periods of time, a duality that has hitherto complicated analysis of this versatile cohesin subunit.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , CoesinasRESUMO
Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin. Our results reveal a "releasing" activity inherent to cohesin complexes transiently associated with Wapl that catalyzes their dissociation from chromosomes. Fusion of Smc3's nucleotide binding domain to α-kleisin's N-terminal domain also reduces cohesin turnover within pericentric chromatin and permits establishment of Wapl-resistant cohesion in the absence of Eco1. We suggest that releasing activity opens the Smc3/α-kleisin interface, creating a DNA exit gate distinct from its proposed entry gate at the Smc1/3 interface. According to this notion, the function of Smc3 acetylation is to block its dissociation from α-kleisin. The functional implications of regulated ring opening are discussed.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Acetiltransferases/metabolismo , Cromossomos Fúngicos/metabolismo , Replicação do DNA , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/citologia , CoesinasRESUMO
Recent data indicate an unexpected requirement for proteins that were hitherto considered to be dedicated to DNA repair to facilitate the faithful disjunction of sister chromatids in anaphase. These include the Bloom's syndrome gene product, BLM and its partners, as well as a number of proteins that are important for preventing Fanconi anemia (FA) in man. As part of an analysis of the roles of these proteins in mitosis, we identified a novel class of anaphase bridge structure, called an ultra-fine anaphase bridge (UFB). These UFBs are also defined by the presence of a SNF2 family protein called PICH. In this review, we will discuss the possible sources of UFBs, and how the BLM, PICH and FA proteins might serve to process these structures in order to maintain genome stability.
Assuntos
Anáfase/genética , Humanos , Modelos GenéticosRESUMO
Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.
Assuntos
Núcleo Celular/metabolismo , Cromossomos/ultraestrutura , Replicação do DNA , DNA/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Ciclo Celular , Linhagem Celular Tumoral , Cromatina/metabolismo , DNA/metabolismo , Dano ao DNA , DNA Helicases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53RESUMO
BACKGROUND: The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an α-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates. RESULTS: In this study, we find transition state mutant versions (Smc1E1158Q and SmcE1155Q) defective in disengagement of their nucleotide binding domains (NBDs), unlike functional cohesin, colocalize with Scc2/4 at core centromeres, sites that catalyze wild-type cohesin's recruitment to sequences 20 kb or more away. In addition to Scc2/4, the unstable association of transition state complexes with core centromeres requires Scc1's association with Smc1 and Smc3 NBDs, ATP-driven NBD engagement, cohesin's Scc3 subunit, and its hinge domain. CONCLUSION: We propose that cohesin's association with chromosomes is driven by two key events. NBD engagement driven by ATP binding produces an unstable association with specific loading sites like core centromeres, whereas subsequent ATP hydrolysis triggers DNA entrapment, which permits translocation along chromatin fibers.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Divisão Celular/fisiologia , Cromatina , Proteínas Cromossômicas não Histona/genética , Hidrólise , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
Several inherited syndromes in humans are associated with cancer predisposition. The gene products defective in two of these disorders, BLM (a helicase defective in Bloom's syndrome) and FANC A-N (defective in Fanconi anaemia), associate in a multienzyme complex called BRAFT. How these proteins suppress tumorigenesis remains unclear, although both conditions are associated with chromosome instability. Here we show that the Fanconi anaemia proteins FANCD2 and FANCI specifically associate with common fragile site loci irrespective of whether the chromosome is broken. Unexpectedly, these loci are frequently interlinked through BLM-associated ultra-fine DNA bridges (UFBs) even as cells traverse mitosis. Similarly to fragile site expression, fragile site bridging is induced after partial inhibition of DNA replication. We propose that, after replication stress, sister chromatids are interlinked by replication intermediates primarily at genetic loci with intrinsic replication difficulties, such as fragile sites. In Bloom's syndrome cells, inefficient resolution of DNA linkages at fragile sites gives rise to increased numbers of anaphase UFBs and micronuclei containing fragile site DNA. Our data have general implications concerning the contribution of fragile site loci to chromosomal instability and tumorigenesis.