RESUMEN

In C. elegans RNA polymerase II (RNAPII) transcription is globally repressed as oocytes prepare for meiosis. Recent work has identified the transcriptional repressors responsible for genome silencing in oocytes, and they are topoisomerase II ( TOP-2 ), condensin II, the H3K9 methyltransferase SET-25 and MET-2 , and the PIE-1 protein. Here, we focus on TOP-2 , condensin II, and MET-2 and ask if they play a similar role during spermatogenesis. We report that spermatocytes undergo transcriptional repression, as inferred by a deactivation of RNAPII, and this requires TOP-2 , the CAPG-2 subunit of condensin II, and the histone methyltransferase MET-2 .

RESUMEN

While it has been appreciated for decades that prophase-arrested oocytes are transcriptionally silenced on a global level, the molecular pathways that promote silencing have remained elusive. Previous work in C. elegans has shown that both topoisomerase II (TOP-2) and condensin II collaborate with the H3K9me heterochromatin pathway to silence gene expression in the germline during L1 starvation, and that the PIE-1 protein silences the genome in the P-lineage of early embryos. Here, we show that all three of these silencing systems, TOP-2/condensin II, H3K9me, and PIE-1, are required for transcriptional repression in oocytes. We find that H3K9me3 marks increase dramatically on chromatin during silencing, and that silencing is under cell cycle control. We also find that PIE-1 localizes to the nucleolus just prior to silencing, and that nucleolar dissolution during silencing is dependent on TOP-2/condensin II. Our data identify both the molecular components and the trigger for genome silencing in oocytes and establish a link between PIE-1 nucleolar residency and its ability to repress transcription.

Asunto(s)

Caenorhabditis elegans , Oocitos , Animales , Caenorhabditis elegans/genética , Cromatina/genética , Cromatina/metabolismo , Células Germinativas/metabolismo , Heterocromatina/metabolismo

RESUMEN

The TOPBP1 and NBS1 proteins are key components of DNA repair and DNA-based signaling systems. TOPBP1 is a multi-BRCT domain containing protein that plays important roles in checkpoint signaling, DNA replication, and DNA repair. Likewise, NBS1, which is a component of the MRE11-RAD50-NBS1 (MRN) complex, functions in both checkpoint signaling and DNA repair. NBS1 also contains BRCT domains, and previous works have shown that TOPBP1 and NBS1 interact with one another. In this work we examine the interaction between TOPBP1 and NBS1 in detail. We report that NBS1 uses its BRCT1 domain to interact with TOPBP1's BRCT1 domain and, separately, with TOPBP1's BRCT2 domain. Thus, NBS1 can make two distinct contacts with TOPBP1. We report that recombinant TOPBP1 and NBS1 proteins bind one another in a purified system, showing that the interaction is direct and does not require post-translational modifications. Surprisingly, we also report that intact BRCT domains are not required for these interactions, as truncated versions of the domains are sufficient to confer binding. For TOPBP1, we find that small 24-29 amino acid sequences within BRCT1 or BRCT2 allow binding to NBS1, in a transferrable manner. These data expand our knowledge of how the crucial DNA damage response proteins TOPBP1 and NBS1 interact with one another and set the stage for functional analysis of the two disparate binding sites for NBS1 on TOPBP1.

Asunto(s)

Enzimas Reparadoras del ADN , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Núcleo Celular/metabolismo , Replicación del ADN , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteína Homóloga de MRE11/metabolismo , Fosforilación

RESUMEN

Ataxia Telangiectasia mutated and RAD3-related (ATR) kinase is activated by DNA replication stress and also by various forms of DNA damage, including DNA double-strand breaks (DSBs). Recruitment to sites of damage is insufficient for ATR activation as one of two known ATR activators, either topoisomerase II-binding protein (TOPBP1) or Ewing's tumor-associated antigen 1, must also be present for signaling to initiate. Here, we employ our recently established DSB-mediated ATR activation in Xenopus egg extract (DMAX) system to examine how TOPBP1 is recruited to DSBs, so that it may activate ATR. We report that TOPBP1 is only transiently present at DSBs, with a half-life of less than 10 minutes. We also examined the relationship between TOPBP1 and the MRE11-RAD50-NBS1 (MRN), CtBP interacting protein (CtIP), and Ataxia Telangiectasia mutated (ATM) network of proteins. Loss of MRN prevents CtIP recruitment to DSBs, and partially inhibits TOPBP1 recruitment. Loss of CtIP has no impact on either MRN or TOPBP1 recruitment. Loss of ATM kinase activity prevents CtIP recruitment and enhances MRN and TOPBP1 recruitment. These findings demonstrate that there are MRN-dependent and independent pathways that recruit TOPBP1 to DSBs for ATR activation. Lastly, we find that both the 9-1-1 complex and MDC1 are dispensable for TOPBP1 recruitment to DSBs.

Asunto(s)

Ataxia Telangiectasia , Roturas del ADN de Doble Cadena , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína Homóloga de MRE11/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Supresoras de Tumor/genética

RESUMEN

Topoisomerase II Binding Protein 1 (TOPBP1) is an important activator of the DNA damage response kinase Ataxia Telangiectasia and Rad3-related (ATR), although the mechanism by which this activation occurs is not yet known. TOPBP1 contains nine copies of the BRCA1 C-terminal repeat (BRCT) motif, which allows protein-protein and protein-DNA interactions. TOPBP1 also contains an ATR activation domain (AAD), which physically interacts with ATR and its partner ATR-interacting protein (ATRIP) in a manner that stimulates ATR kinase activity. It is unclear which of TOPBP1's nine BRCT domains participate in the reaction, as well as the individual roles played by these relevant BRCT domains. To address this knowledge gap, here, we delineated a minimal TOPBP1 that can activate ATR at DNA double-strand breaks in a regulated manner. We named this minimal TOPBP1 "Junior" and we show that Junior is composed of just three regions: BRCT0-2, the AAD, and BRCT7&8. We further defined the individual functions of these three regions by showing that BRCT0-2 is required for recruitment to DNA double-strand breaks and is dispensable thereafter, and that BRCT7&8 is dispensable for recruitment but essential to allow the AAD to multimerize and activate ATR. The delineation of TOPBP1 Junior creates a leaner, simplified, and better understood TOPBP1 and provides insight into the mechanism of ATR activation.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Proteínas Nucleares , Proteínas de Xenopus , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Xenopus , Proteínas de Xenopus/metabolismo

RESUMEN

While much is known about how transcription is controlled at individual genes, comparatively little is known about how cells regulate gene expression on a genome-wide level. Here, we identify a molecular pathway in the C. elegans germline that controls transcription globally in response to nutritional stress. We report that when embryos hatch into L1 larvae, they sense the nutritional status of their environment, and if food is unavailable, they repress gene expression via a global chromatin compaction (GCC) pathway. GCC is triggered by the energy-sensing kinase AMPK and is mediated by a novel mechanism that involves the topoisomerase II/condensin II axis acting upstream of heterochromatin assembly. When the GCC pathway is inactivated, then transcription persists during starvation. These results define a new mode of whole-genome control of transcription.

Asunto(s)

Caenorhabditis elegans/genética , Cromatina/química , Regulación del Desarrollo de la Expresión Génica , Genoma de los Helmintos , Proteínas Quinasas/genética , Inanición/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Embrión no Mamífero , Histona Acetiltransferasas/antagonistas & inhibidores , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Óvulo/metabolismo , Proteínas Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Espermatozoides/metabolismo , Inanición/metabolismo , Transcripción Genética

RESUMEN

The protein kinase ATR is activated at sites of DNA double-strand breaks where it plays important roles in promoting DNA end resection and regulating cell cycle progression. TOPBP1 is a multi BRCT repeat containing protein that activates ATR at DSBs. Here we have developed an experimental tool, the DMAX system, to study the biochemical mechanism for TOPBP1-mediated ATR signalling. DMAX combines simple, linear dsDNA molecules with Xenopus egg extracts and results in a physiologically relevant, DSB-induced activation of ATR. We find that DNAs of 5000 nucleotides, at femtomolar concentration, potently activate ATR in this system. By combining immunodepletion and add-back of TOPBP1 point mutants we use DMAX to determine which of TOPBP1's nine BRCT domains are required for recruitment of TOPBP1 to DSBs and which domains are needed for ATR-mediated phosphorylation of CHK1. We find that BRCT1 and BRCT7 are important for recruitment and that BRCT5 functions downstream of recruitment to promote ATR-mediated phosphorylation of CHK1. We also show that BRCT7 plays a second role, independent of recruitment, in promoting ATR signalling. These findings supply a new research tool for, and new insights into, ATR biology.

Asunto(s)

Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Óvulo , Transducción de Señal/genética , Transducción de Señal/fisiología , Extractos de Tejidos , Xenopus , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , ADN/genética , ADN/metabolismo , Fosforilación/genética

RESUMEN

TOPBP1 is an important scaffold protein that helps orchestrate the cellular response to DNA damage. Although it has been previously appreciated that TOPBP1 can form oligomers, how this occurs and the functional consequences for oligomerization were not yet known. Here, we use protein binding assays and other biochemical techniques to study how TOPBP1 self associates. TOPBP1 contains 9 copies of the BRCT domain, and we report that a subset of these BRCT domains interact with one another to drive oligomerization. An intact BRCT 2 domain is required for TOPBP1 oligomerization and we find that the BRCT1&2 region of TOPBP1 interacts with itself and with the BRCT4&5 pair. RAD9 and RHINO are two heterologous binding partners for TOPBP1's BRCT 1&2 domains, and we show that binding of these partners does not come at the expense of TOPBP1 oligomerization. Furthermore, we show that a TOPBP1 oligomer can simultaneously interact with both RAD9 and RHINO. Lastly, we find that the oligomeric state necessary for TOPBP1 to activate the ATR protein kinase is likely to be a tetramer.

Asunto(s)

Daño del ADN , Proteínas de Unión al ADN/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , Transducción de Señal , Xenopus/genética , Xenopus/metabolismo

RESUMEN

In Caenorhabditis elegans, the primordial germ cells Z2 and Z3 are born during early embryogenesis and then held in a transcriptionally quiescent state where the genome is highly compacted. When hatched L1s feed, the germline genome decompacts, and RNAPII is abruptly and globally activated. A previously documented yet unexplained feature of germline genome activation in the worm is the appearance of numerous DNA breaks coincident with RNAPII transcription. Here, we show that the DNA breaks are induced by topoisomerase II and that they function to recruit the RUVB complex to chromosomes so that RUVB can decompact the chromatin. DNA break- and RUVB-mediated decompaction is required for zygotic genome activation. This work highlights the importance of global chromatin decompaction in the rapid induction of gene expression and shows that one way cells achieve global decompaction is through programmed DNA breaks.

Asunto(s)

Roturas del ADN de Doble Cadena , Daño del ADN/genética , Desarrollo Embrionario/genética , Células Germinativas/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/metabolismo , Cromosomas/metabolismo , Reparación del ADN/fisiología , Mutación/genética , Cigoto/metabolismo

RESUMEN

Plasmid engineering and molecular cloning is a virtually ubiquitous tool in biology. Although various methods have been developed for ligating DNA molecules or targeted mutagenesis of plasmids, each has its limitations. Many of the commonly used laboratory strategies are inefficient, while commercially available kits are quite costly and often specialized for highly specific circumstances. Here, we describe the SapI/AarI incision mediated plasmid editing (SIMPLE) method, which allows users to perform site-directed mutagenesis, deletions, and even short insertions into any plasmid in a single PCR reaction, using just one restriction enzyme. In addition, the SIMPLE method can be adapted to insert any sized DNA fragment into a vector using a two-step PCR approach, and can be used to ligate any number of DNA fragments with non-compatible ends in the specific order desired. The SIMPLE method provides researches an efficient and powerful tool with a broad range of applications for molecular cloning.

Asunto(s)

Edición Génica/métodos , Plásmidos/genética , Clonación Molecular , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo

RESUMEN

To better understand how the cellular response to DNA replication stress is regulated during embryonic development, we and others have established the early C. elegans embryo as a model system to study this important problem. As is the case in most eukaryotic cell types, the replication stress response is controlled by the ATR kinase in early worm embryos. In this report we use RNAi to systematically characterize ATR pathway components for roles in promoting cell cycle delay during a replication stress response, and we find that these genetic requirements vary, depending on the source of stress. We also examine how individual cell types within the embryo respond to replication stress, and we find that the strength of the response, as defined by duration of cell cycle delay, varies dramatically within blastomeres of the early embryo. Our studies shed light on how the replication stress response is managed in the context of embryonic development and show that this pathway is subject to developmental regulation.

Asunto(s)

Caenorhabditis elegans/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/antagonistas & inhibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/metabolismo , Embrión no Mamífero/efectos de la radiación , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Embrionario/efectos de la radiación , Hidroxiurea/toxicidad , Interfase/efectos de los fármacos , Interfase/efectos de la radiación , Interferencia de ARN , Rayos Ultravioleta

RESUMEN

In early C. elegans embryos the timing of cell division is both invariant and developmentally regulated, yet how the cell cycle is controlled in the embryo and how cell cycle timing impacts early development remain important, unanswered questions. Here, I focus on the cyclin B3 ortholog CYB-3, and show that this cyclin has the unusual property of controlling both the timely progression through S-phase and mitotic entry, suggesting that CYB-3 is both an S-phase-promoting and mitosis-promoting factor. Furthermore, I find that CYB-3 is asymmetrically distributed in the two-cell embryo, such that the somatic precursor AB cell contains ∼2.5-fold more CYB-3 than its sister cell, the germline progenitor P1 CYB-3 is not only physically limited in P1 but also functionally limited, and this asymmetry is controlled by the par polarity network. These findings highlight the importance of the CYB-3 B3-type cyclin in cell cycle regulation in the early embryo and suggest that CYB-3 asymmetry helps establish the well-documented cell cycle asynchrony that occurs during cell division within the P-lineage.

Asunto(s)

Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Ciclina B/metabolismo , Embrión no Mamífero/metabolismo , Mitosis/fisiología , Fase S/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Ciclina B/genética , Embrión no Mamífero/citología , Mitosis/genética , Fase S/genética

RESUMEN

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT (BRCA1 C terminus) repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation; however, the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated single-stranded DNA. We identify a point mutant that abrogates this interaction and show that this mutant fails to accumulate at sites of DNA damage and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

Asunto(s)

Proteínas Portadoras/química , ADN de Cadena Simple/química , Proteína de Replicación A/química , Animales , Daño del ADN , Proteínas de Unión al ADN , Unión Proteica , Estructura Terciaria de Proteína , Xenopus laevis

RESUMEN

Recent findings have identified highly transcribed genes as a source of genome instability; however, the degree to which large-scale shifts in transcriptional activity cause DNA damage was not known. One example of a large-scale shift in transcriptional activity occurs during development, when maternal regulators are destroyed and zygotic genome activation (ZGA) occurs. Here, we show that ZGA triggers widespread chromosome damage in the primordial germ cells of the nematode C. elegans. We show that ZGA-induced DNA damage activates a checkpoint response, the damage is repaired by factors required for inter-sister homologous recombination, and topoisomerase II plays a role in generating the damage. These findings identify ZGA as a source of intrinsic genome instability in the germline and suggest that genome destabilization may be a general consequence of extreme shifts in cellular transcriptional load.

Asunto(s)

Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/citología , Puntos de Control del Ciclo Celular/genética , Daño del ADN/genética , Inestabilidad Genómica/genética , Células Germinativas/citología , Cigoto/citología , Animales , Caenorhabditis elegans/genética , Mutación/genética

RESUMEN

Recent work from our laboratory has identified zygotic genome activation as a source of intrinsic DNA damage during germline development in C. elegans. More specifically, we have found that the primordial germ cells Z2 and Z3 experience DNA damage and damage checkpoint activation shortly after RNA polymerase II is activated by a nutrient-dependent signal in L1 stage animals. In this Commentary we review these data, put them into context with other examples of programmed DNA double-strand breaks (DSBs) during gene activation, and speculate as to how a DSB would facilitate signal-dependent activation of gene expression.

RESUMEN

TopBP1, a multiple-BRCT-containing protein, plays diverse functions in DNA metabolism including DNA replication, DNA damage response and transcriptional regulation. The cytoplasmic localization of TopBP1 has been found to be associated with breast cancer susceptibility in clinical studies, suggesting the biological significance of TopBP1's sub-cellular localization. However, it remains elusive how TopBP1 is shuttled into nucleus and recruited to chromatin under normal or stressful conditions. Taking advantage of Xenopus egg extract, we identified Importin ß as a new interacting protein of the TopBP1 C-terminus. We verified the TopBP1-Importin ß association via GST pulldown and coimmunoprecipitation assays. We then demonstrated that TopBP1's C-terminal motif (designated as CTM, 23 amino acids) containing a putative NLS (nuclear localization signal) was required for Importin ß interaction and that CT100 of Importin ß (100 amino acids of extreme C-terminus of Importin ß) was required for TopBP1 interaction. Further structure-function analysis reveals that the CTM of TopBP1 is essential for TopBP1's nuclear import and subsequent chromatin recruitment, thereby playing important roles in DNA replication and mitomycin C (MMC)-induced Chk1 phosphorylation. In addition, Importin ß-specific inhibitor importazole inhibits TopBP1's nuclear import and the MMC-induced Chk1 phosphorylation. With ongoing DNA replication, the Importin ß-dependent nuclear import of TopBP1 was indeed required for the MMC-induced Chk1 phosphorylation. Our data also suggest that checkpoint activation requires more TopBP1 than DNA replication does. The requirement of TopBP1's CTM motif for ATR-Chk1 checkpoint can be bypassed in a nucleus-free AT70 system. Taken together, our findings suggest that the CTM motif-mediated TopBP1 shuttling into nucleus via Importin ß plays an important role in the ATR-Chk1 checkpoint signaling in Xenopus egg extracts.

Asunto(s)

Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Proteínas Quinasas/metabolismo , Xenopus laevis/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Reparación del ADN , Proteínas de Unión al ADN , Mitomicina/farmacología , Datos de Secuencia Molecular , Óvulo/metabolismo , Fosforilación/efectos de los fármacos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas de Xenopus , Xenopus laevis/crecimiento & desarrollo , beta Carioferinas/antagonistas & inhibidores

RESUMEN

The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.

Asunto(s)

Replicación del ADN , Proteína Fosfatasa 2/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Extractos Celulares , Núcleo Celular/genética , Núcleo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Citoplasma/genética , Citoplasma/metabolismo , Femenino , Immunoblotting , Masculino , Óvulo/citología , Óvulo/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2/genética , Subunidades de Proteína/metabolismo , Fase S , Transducción de Señal , Proteínas de Xenopus/genética , Xenopus laevis/genética

RESUMEN

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-delta, and Pol-epsilon, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer-template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

Asunto(s)

Cartilla de ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple/metabolismo , Animales , Afidicolina/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cromatina/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , Cartilla de ADN/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN , Inhibidores Enzimáticos/metabolismo , Femenino , Masculino , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo

RESUMEN

In this issue of Molecular Cell, Ward et al. (2010) identify two genes whose products act redundantly to clear Rad51 from DNA after successful strand invasion, thereby enabling the downstream events of homologous recombination to go smoothly.

Asunto(s)

Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/enzimología , Reparación del ADN/fisiología , ADN de Helmintos/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Meiosis , Modelos Genéticos