RESUMEN
The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.
RESUMEN
RecA protein and RecA/Rad51 orthologues are required for homologous recombination and DNA repair in all living creatures. RecA/Rad51 catalyzes formation of the D-loop, an obligatory recombination intermediate, through an ATP-dependent reaction consisting of two phases: homology recognition between double-stranded (ds)DNA and single-stranded (ss)DNA to form a hybrid-duplex core of 6-8 base pairs and subsequent hybrid-duplex/D-loop processing. How dsDNA recognizes homologous ssDNA is controversial. The aromatic residue at the tip of the ß-hairpin loop (L2) was shown to stabilize dsDNA-strand separation. We tested a model in which dsDNA strands were separated by the aromatic residue before homology recognition and found that the aromatic residue was not essential to homology recognition, but was required for D-loop processing. Contrary to the model, we found that the double helix was not unwound even a single turn during search for sequence homology, but rather was unwound only after the homologous sequence was recognized. These results suggest that dsDNA recognizes its homologous ssDNA before strand separation. The search for homologous sequence with homologous ssDNA without dsDNA-strand separation does not generate stress within the dsDNA; this would be an advantage for dsDNA to express homology-dependent functions in vivo and also in vitro.
Asunto(s)
ADN de Cadena Simple , Recombinación Homóloga , Recombinasa Rad51 , Emparejamiento Base , ADN/química , ADN de Cadena Simple/genética , Rec A Recombinasas/metabolismoRESUMEN
DNA replication is central to cell proliferation. Studies in the past six decades since the proposal of a semiconservative mode of DNA replication have confirmed the high degree of conservation of the basic machinery of DNA replication from prokaryotes to eukaryotes. However, the need for replication of a substantially longer segment of DNA in coordination with various internal and external signals in eukaryotic cells has led to more complex and versatile regulatory strategies. The replication program in higher eukaryotes is under a dynamic and plastic regulation within a single cell, or within the cell population, or during development. We review here various regulatory mechanisms that control the replication program in eukaryotes and discuss future directions in this dynamic field.
Asunto(s)
Cromosomas/metabolismo , Replicación del ADN , Animales , Humanos , Origen de Réplica , Fase SRESUMEN
The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7Fl/Fl NestinCre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7Fl/F l NestinCre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinasas , Animales , Ratones , Proteínas de Ciclo Celular/metabolismo , Cerebelo/metabolismo , Replicación del ADN , Nestina/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Inflammation is implicated in the onset and progression of various diseases, including cerebral pathologies. Here, we report that DJ-1, which plays a role within cells as an antioxidant protein, functions as a damage-associated molecular pattern (DAMP) and triggers inflammation if released from dead cells into the extracellular space. We first found that recombinant DJ-1 protein induces the production of various inflammatory cytokines in bone marrow-derived macrophages (BMMs) and dendritic cells (BMDCs). We further identified a unique peptide sequence in the αG and αH helices of DJ-1 that activates Toll-like receptor 2 (TLR2) and TLR4. In the ischemic brain, DJ-1 is released into the extracellular space from necrotic neurons within 24 h after stroke onset and makes direct contact with TLR2 and TLR4 in infiltrating myeloid cells. Although DJ-1 deficiency in a murine model of middle cerebral artery occlusion did not attenuate neuronal injury, the inflammatory cytokine expression in infiltrating immune cells was significantly decreased. Next, we found that the administration of an antibody to neutralize extracellular DJ-1 suppressed cerebral post-ischemic inflammation and attenuated ischemic neuronal damage. Our results demonstrate a previously unknown function of DJ-1 as a DAMP and suggest that extracellular DJ-1 could be a therapeutic target to prevent inflammation in tissue injuries and neurodegenerative diseases.
Asunto(s)
Isquemia Encefálica/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Alarminas/metabolismo , Animales , Encéfalo/metabolismo , Isquemia Encefálica/fisiopatología , Citocinas/inmunología , Modelos Animales de Enfermedad , Infarto de la Arteria Cerebral Media/inmunología , Infarto de la Arteria Cerebral Media/patología , Inflamación , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Proteína Desglicasa DJ-1/fisiología , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/fisiopatología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.
Asunto(s)
Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia , Poli(ADP-Ribosa) Polimerasa-1 , Hipoxia de la Célula , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Receptor Notch3/metabolismoRESUMEN
The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.
Asunto(s)
Ciclo Celular , Técnicas Citológicas , Animales , Células COS , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Fluorescencia , Geminina , Células HeLa , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Transgénicos , Microscopía Confocal , Datos de Secuencia Molecular , Morfogénesis , Neoplasias/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , UbiquitinaciónRESUMEN
Rif1 regulates DNA replication timing and double-strand break repair, and its depletion induces transcriptional bursting of two-cell (2C) zygote-specific genes in mouse ES cells. However, how Rif1 regulates zygotic transcription is unclear. We show here that Rif1 depletion promotes the formation of a unique Zscan4 enhancer structure harboring both histone H3 lysine 27 acetylation (H3K27ac) and moderate levels of silencing chromatin mark H3K9me3. Curiously, another enhancer mark H3K4me1 is missing, whereas DNA methylation is still maintained in the structure, which spreads across gene bodies and neighboring regions within the Zscan4 gene cluster. We also found by function analyses of Rif1 domains in ES cells that ectopic expression of Rif1 lacking N-terminal domain results in upregulation of 2C transcripts. This appears to be caused by dominant negative inhibition of endogenous Rif1 protein localization at the nuclear periphery through formation of hetero-oligomers between the N-terminally truncated and endogenous forms. Strikingly, in murine 2C embryos, most of Rif1-derived polypeptides are expressed as truncated forms in soluble nuclear or cytosolic fraction and are likely nonfunctional. Toward the morula stage, the full-length form of Rif1 gradually increased. Our results suggest that the absence of the functional full-length Rif1 due to its instability or alternative splicing and potential inactivation of Rif1 through dominant inhibition by N-terminally truncated Rif1 polypeptides may be involved in 2C-specific transcription program.
Asunto(s)
Replicación del ADN/fisiología , Proteínas de Unión a Telómeros/fisiología , Activación Transcripcional/fisiología , Cigoto/metabolismo , Acetilación , Animales , Cromatina/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Nearly 70 years after the proposal of semiconservative replication of generic material by Watson and Crick, we now understand many of the proteins involved in the replication of host chromosomes and how they operate. The initiator and replicator, proposed in the replicon hypothesis, are now well defined in both prokaryotes and eukaryotes. On the other hand, studies in prokaryotes and Archaea indicate alternative modes of initiation, which may not depend on an initiator. Here I summarize recent progress in the field of DNA replication and discuss the evolution of replication systems.
Asunto(s)
Replicación del ADN , Origen de Réplica , Escherichia coli/metabolismo , Proteínas de Unión al ADN/metabolismo , Replicón , Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos , ADN Bacteriano/genéticaRESUMEN
NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.
Asunto(s)
Replicación del ADN , Coactivadores de Receptor Nuclear/fisiología , Origen de Réplica , Animales , Células Cultivadas , Senescencia Celular , Células HeLa , Humanos , Ratones , Componente 7 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Técnicas del Sistema de Dos Híbridos , Xenopus laevisRESUMEN
Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7-ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7-ASK (Dbf4) complex in a manner dependent on ATR-Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C(Cdh1) through degradation of Cdh1 upon replication block, thereby stabilizing APC/C(Cdh1) substrates, including Cdc7-ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin-proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Replicación del ADN , Antígenos CD , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Cadherinas/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Estabilidad de Enzimas , Genes APC/fisiología , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de SeñalRESUMEN
G-quadruplex is one of the best-studied non-B type DNA that is now known to be prevalently present in the genomes of almost all the biological species. Recent studies reveal roles of G-quadruplex (G4) structures in various nucleic acids and chromosome transactions. In this short article, we will first describe recent findings on the roles of G4 in regulation of DNA replication. G4 is involved in regulation of spatio-temporal regulation of DNA replication through interaction with a specific binding protein, Rif1. This regulation is at least partially mediated by generation of specific chromatin architecture through Rif1-G4 interactions. We will also describe recent studies showing the potential roles of G4 in initiation of DNA replication. Next, we will present showcases of highly diversified roles of DNA G4 and RNA G4 in regulation of nucleic acid and chromosome functions. Finally, we will discuss how the formation of cellular G4 could be regulated.
Asunto(s)
Replicación del ADN , ADN/genética , G-Cuádruplex , ARN/genética , Animales , ADN/química , Epigénesis Genética , Humanos , ARN/química , Transcripción GenéticaRESUMEN
G-quadrupex is now known to play crucial roles in various biological reactions. However, direct evidence for its presence in cells has been limited, due to the lack of versatile and non-biased methodology. We use Rif1 binding sites on the fission yeast genome, which has been shown to adopt G4 structures, as a model to prove that Rif1 BS indeed adopt G4 structure in cells. We take advantage of the presence of a single-stranded loop in the G4 structure. Rif1BS is unique in that they contain unusually long loop sequences, and we replace them with a 18 bp I-SceI restriction site. We show in vitro that I-SceI in the loop is not cleaved when G4 is formed on duplex Rif1BS DNA, but is cleaved when G4 is not formed due to a mutation in the G-tracts. This is observed both heat-induced and transcription-induced G4 structure, and gives proof of evidence for this procedure. We apply this strategy for detection of a G4 structure at the same Rif1BS in fission yeast cells. We present evidence that in vivo cleavage of I-SceI can be a measure for the presence of G4 at the target sequence in cells as well. The method described here gives a platform strategy for genome-wide analyses of cellular G4 and their dynamic formation and disruption.
Asunto(s)
G-Cuádruplex , Genoma Fúngico , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/metabolismo , Sitios de Unión , Desnaturalización de Ácido Nucleico , Schizosaccharomyces/metabolismoRESUMEN
Higher-order chromosome structure is assumed to control various DNA-templated reactions in eukaryotes. Meiotic chromosomes implement developed structures called "axes" and "loops"; both are suggested to tether each other, activating Spo11 to catalyze meiotic DNA double-strand breaks (DSBs) at recombination hotspots. We found that the Schizosaccharomyces pombe Spo11 homolog Rec12 and its partners form two distinct subcomplexes, DSBC (Rec6-Rec12-Rec14) and SFT (Rec7-Rec15-Rec24). Mde2, whose expression is strictly regulated by the replication checkpoint, interacts with Rec15 to stabilize the SFT subcomplex and further binds Rec14 in DSBC. Rec10 provides a docking platform for SFT binding to axes and can partially interact with DSB sites located in loops depending upon Mde2, which is indicative of the formation of multiprotein-based tethered axis-loop complex. These data lead us to propose a mechanism by which Mde2 functions as a recombination initiation mediator to tether axes and loops, in liaison with the meiotic replication checkpoint.
Asunto(s)
Cromosomas/metabolismo , Endodesoxirribonucleasas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Recombinación Genética , Fase S , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Roturas del ADN de Doble Cadena , Meiosis/genética , Schizosaccharomyces/genéticaRESUMEN
The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Elementos Aisladores/genética , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/fisiología , Secuencia de Bases , Replicación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Eucromatina/ultraestructura , Heterocromatina/ultraestructura , Ensayos Analíticos de Alto Rendimiento , Origen de RéplicaRESUMEN
One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1Δ, a null mutant of rif1(+), a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1Δ in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1Δ. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes.
Asunto(s)
Momento de Replicación del ADN/genética , Origen de Réplica/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/fisiología , Centrómero/metabolismo , Replicación del ADN/genética , Fase G1 , Eliminación de Gen , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Telómero/metabolismo , Proteínas de Unión a Telómeros/genéticaRESUMEN
Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.
Asunto(s)
G-Cuádruplex , Modelos Moleculares , Proteínas de Unión a Telómeros/metabolismo , Animales , Sitios de Unión , Cromatografía en Gel , Dimerización , Células HEK293 , Humanos , Inmunoprecipitación , Cinética , Ratones , Conformación de Ácido Nucleico , Tamaño de la Partícula , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/aislamiento & purificaciónRESUMEN
G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.
Asunto(s)
ADN de Hongos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Unión a Telómeros/química , Secuencia de Bases , Sitios de Unión , Huella de ADN , Replicación del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , G-Cuádruplex , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Schizosaccharomyces/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Unión a Telómeros/genética , Proteínas de Unión a Telómeros/metabolismoRESUMEN
Mini-chromosome maintenance (Mcm) is a central component for DNA unwinding reaction during eukaryotic DNA replication. Mcm2â¼7, each containing a conserved ATPase motif, form a six subunit-heterohexamer. Although the reconstituted Mcm2â¼7-Cdc45-GINS (CMG) complex displays DNA unwinding activity, the Mcm2â¼7 complex does not generally exhibit helicase activity under a normal assay condition. We detected a strong DNA strand annealing activity in the purified mouse Mcm2â¼7 heterohexamer, which promotes rapid reassociation of displaced complementary single-stranded DNAs, suggesting a potential cause for its inability to exhibit DNA helicase activity. Indeed, DNA unwinding activity of Mcm2â¼7 could be detected in the presence of a single-stranded DNA that is complementary to the displaced strand, which would prevent its reannealing to the template. ATPase-deficient mutations in Mcm2, 4, 5 and 6 subunits inactivated the annealing activity, while those in Mcm2 and 5 subunits alone did not. The annealing activity of Mcm2â¼7 does not require Mg2+ and ATP, and is adversely inhibited by the presence of high concentration of Mg2+ and ATP while activated by similar concentrations of ADP. Our findings show that the DNA helicase activity of Mcm2â¼7 may be masked by its unexpectedly strong annealing activity, and suggest potential physiological roles of strand annealing activity of Mcm during replication stress responses.
Asunto(s)
Replicación del ADN , ADN/química , Proteínas de Mantenimiento de Minicromosoma/fisiología , Adenosina Trifosfato/química , Animales , Células Cultivadas , Hidrólisis , Insectos , Cinética , Ratones , Proteínas de Mantenimiento de Minicromosoma/química , Fosforilación , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Members of the BTB-ZF transcription factor family play important roles in lymphocyte development. During T cell development, ZNF131, a BTB-ZF protein, is critical for the double-negative (DN) to double-positive (DP) transition and is also involved in cell proliferation. Here, we report that knockout of Znf131â¯at the pre-pro-B cell stage in mb1-Cre knock-in mouse resulted in defect of pro-B to pre-B cell transition. ZNF131 was shown to be required for efficient pro-B cell proliferation as well as for immunoglobulin heavy chain gene rearrangement that occurs in the proliferating pro-B cells. We speculate that inefficient gene rearrangement may be due to loss of cell proliferation, since cell cycle progression and immunoglobulin gene rearrangement, which would occur in a mutually exclusive manner, may be interconnected or coupled to avoid occurrence of genomic instability. ZNF131 suppresses expression of Cdk inhibitor, p21cip1, and that of pro-apoptotic factors, Bax and Puma, targets of p53, to facilitate cell cycle progression and suppress unnecessary apoptosis, respectively, of pro-B cells. There results demonstrate the essential roles of ZNF131 in coordinating the B cell differentiation and proliferation.