RESUMEN

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Asunto(s)

Proteínas de Ciclo Celular , Geminina , Animales , Geminina/metabolismo , Geminina/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Replicación del ADN , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Dominios Proteicos , Xenopus , Humanos , Proteínas de Unión al ADN

RESUMEN

Fetal bovine serum (FBS) substitution remains one of the challenges to the realization of cultured meat production in the marketplace. In this study, three methods were developed to extract a substitute for FBS using egg white extract (EWE): using 25 mM CaCl2/2.5 % ammonium sulfate/citric acid (A); ethyl alcohol (B); and 5 % ammonium sulfate/citric acid (C). B EWE can effectively replace up to 50 % of FBS in growth media (10 % of the total). Ovalbumin in the extracts can promote cell proliferation, and components along the 12 kDa protein band have the potential to inhibit cell proliferation. Chick primary muscle cells applied with B EWE, an edible material that improved the cost and time efficiency of cultured meat production, effectively proliferated/differentiated. Therefore, EWE extracted using ethyl alcohol may be used as an FBS substitute to reduce animal sacrifices and should be considered a viable alternative to FBS for cultured meat.

RESUMEN

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

Asunto(s)

Microtúbulos , Animales , Xenopus , Xenopus laevis , Ciclo Celular , Citoplasma

RESUMEN

Condensin I is a pentameric protein complex that plays an essential role in mitotic chromosome assembly in eukaryotic cells. Although it has been shown that condensin I loading is mitosis specific, it remains poorly understood how the robust cell cycle regulation of condensin I is achieved. Here, we set up a panel of in vitro assays to demonstrate that cell cycle-specific loading of condensin I is regulated by the N-terminal tail (N-tail) of its kleisin subunit CAP-H. Deletion of the N-tail accelerates condensin I loading and chromosome assembly in Xenopus egg mitotic extracts. Phosphorylation-deficient and phosphorylation-mimetic mutations in the CAP-H N-tail decelerate and accelerate condensin I loading, respectively. Remarkably, deletion of the N-tail enables condensin I to assemble mitotic chromosome-like structures even in interphase extracts. Together with other extract-free functional assays in vitro, our results uncover one of the multilayered mechanisms that ensure cell cycle-specific loading of condensin I onto chromosomes.

Asunto(s)

Adenosina Trifosfatasas , Cromosomas , Ciclo Celular , Cromosomas/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Mitosis , Proteínas de Ciclo Celular/genética

RESUMEN

The nuclear pore complex (NPC) is the conduit in the nuclear envelope through which proteins and RNA are transported between the cytoplasm and nucleus. Xenopus egg extracts that support de novo assembly of nuclei have provided a robust system to study NPC structure and function because the biochemical composition of the extract can be easily manipulated. Here we describe how to assemble nuclei in Xenopus egg extract, how to visualize and analyze NPCs in both live and fixed samples, and different approaches to altering nucleocytoplasmic transport in extract.

Asunto(s)

Membrana Nuclear , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Microscopía/métodos , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Xenopus laevis/metabolismo

RESUMEN

DNA replication is crucial for cell viability and genome integrity. Despite its crucial role in genome duplication, the final stage of DNA replication, which is termed termination, is relatively unexplored. Our knowledge of termination is limited by cellular approaches to study DNA replication, which cannot readily detect termination. In contrast, the Xenopus laevis egg extract system allows for all of DNA replication to be readily detected. Here we describe the use of this system and assays to monitor replication termination.

Asunto(s)

Replicación del ADN , Oocitos , Animales , Genoma , Xenopus laevis/genética

RESUMEN

During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. Here, we show that Nup50 plays a crucial role in NPC assembly independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of Nup50 protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and for NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α/ß, the GTPase Ran, or chromatin is crucial for its function in the assembly process. Instead, an N-terminal fragment of Nup50 can stimulate the Ran GTPase guanine nucleotide exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in NPC reformation at the end of mitosis. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild-type protein in in vitro NPC assembly assays, whereas excess RCC1 can compensate the loss of Nup50.

Asunto(s)

Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Mitosis , Mutación , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/genética , Células HeLa , Humanos , Proteínas de Complejo Poro Nuclear/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Xenopus laevis

RESUMEN

The anaphase promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, is a key regulator of mitotic progression. Upon activation in mitosis, the APC/C targets its two essential substrates, securin and cyclin B, for proteasomal destruction. Cyclin B is the activator of cyclin-dependent kinase 1 (Cdk1), the major mitotic kinase, and both cyclin B and securin are safeguards of sister chromatid cohesion. Conversely, the degradation of securin and cyclin B promotes sister chromatid separation and mitotic exit. The negative feedback loop between Cdk1 and APC/C-Cdk1 activating the APC/C and the APC/C inactivating Cdk1-constitutes the core of the biochemical cell cycle oscillator.Since its discovery three decades ago, the mechanisms of APC /C regulation have been intensively studied, and several in vitro assays exist to measure the activity of the APC /C in different activation states. However, most of these assays require the purification of numerous recombinant enzymes involved in the ubiquitylation process (e.g., ubiquitin, the E1 and E2 ubiquitin ligases, and the APC /C) and/or the use of radioactive isotopes. In this chapter, we describe an easy-to-implement method to continuously measure APC /C activity in Xenopus laevis egg extracts using APC /C substrates fused to fluorescent proteins and a fluorescence plate reader. Because the egg extract provides all important enzymes and proteins for the reaction, this method can be used largely without the need for recombinant protein purification. It can also easily be adapted to test the activity of APC /C mutants or investigate other mechanisms of APC /C regulation.

Asunto(s)

Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclina B/metabolismo , Proteínas Luminiscentes/metabolismo , Securina/metabolismo , Xenopus laevis/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , Ciclina B/genética , Retroalimentación Fisiológica , Femenino , Proteínas Luminiscentes/genética , Mitosis , Imagen Óptica/instrumentación , Óvulo/metabolismo , Proteínas Quinasas/metabolismo , Proteolisis , Proteínas Recombinantes/metabolismo , Securina/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo

RESUMEN

SummaryMuch effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract- co-injected group compared with those in the no egg extract-injected (NT) group (38-66% vs 18-34%, P<0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P<0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.

Asunto(s)

Blastocisto/citología , Extractos Celulares/química , Oocitos/metabolismo , Proteínas de Unión al ARN/metabolismo , Xenopus laevis/metabolismo , Animales , Blastocisto/metabolismo , Factor 1 de Respuesta al Butirato , Clonación de Organismos/métodos , Células del Cúmulo/metabolismo , Desarrollo Embrionario , Femenino , Regulación del Desarrollo de la Expresión Génica , Ratones , Células 3T3 NIH , Técnicas de Transferencia Nuclear , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Oocitos/citología , Proteínas de Unión al ARN/genética

RESUMEN

DNA single-strand breaks (SSBs) are the most common type of DNA lesions as they are generated approximately 10,000 times per mammalian cell each day. Unrepaired SSBs compromise DNA replication and transcription programs, leading to genome instability, and have been implicated in many diseases including cancer. In this chapter, we introduce methods to study the ATR-Chk1 DNA damage response (DDR) pathway and DNA repair pathway in response to a site-specific, defined SSB plasmid in Xenopus laevis egg extracts. This experimental system can be applied in future studies to reveal many aspects of the molecular mechanisms of SSB repair and signaling in eukaryotes.

Asunto(s)

Roturas del ADN de Cadena Simple , Reparación del ADN , ADN/genética , Xenopus laevis/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , ADN/metabolismo , Oocitos , Plásmidos/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo

RESUMEN

Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.

Asunto(s)

ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Reparación del ADN/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple , Proteínas de Unión al ADN/fisiología , Femenino , Masculino , Proteolisis , Imagen Individual de Molécula/métodos , Xenopus laevis/metabolismo

RESUMEN

Coordination between actin filaments and microtubules is critical to complete important steps during cell division. For instance, cytoplasmic actin filament dynamics play an active role in the off-center positioning of the spindle during metaphase I in mouse oocytes [1-3] or in gathering the chromosomes to ensure proper spindle formation in starfish oocytes [4, 5], whereas cortical actin filaments control spindle rotation and positioning in adherent cells or in mouse oocytes [6-9]. Several molecular effectors have been found to facilitate anchoring between the meiotic spindle and the cortical actin [10-14]. In vitro reconstitutions have provided detailed insights in the biochemical and physical interactions between microtubules and actin filaments [15-20]. Yet how actin meshwork architecture affects microtubule dynamics is still unclear. Here, we reconstituted microtubule aster in the presence of a meshwork of actin filaments using confined actin-intact Xenopus egg extracts. We found that actin filament branching reduces the lengths and growth rates of microtubules and constrains the mobility of microtubule asters. By reconstituting the interaction between dynamic actin filaments and microtubules in a minimal system based on purified proteins, we found that the branching of actin filaments is sufficient to block microtubule growth and trigger microtubule disassembly. In a further exploration of Xenopus egg extracts, we found that dense and static branched actin meshwork perturbs monopolar spindle assembly by constraining the motion of the spindle pole. Interestingly, monopolar spindle assembly was not constrained in conditions supporting dynamic meshwork rearrangements. We propose that branched actin filament meshwork provides physical barriers that limit microtubule growth.

Asunto(s)

Citoesqueleto de Actina/fisiología , Actinas/fisiología , Microtúbulos/fisiología , Oocitos/fisiología , Xenopus laevis/fisiología , Animales

RESUMEN

Egg extracts of the African clawed frog Xenopus laevis have provided a cell-free system instrumental in elucidating events of the cell cycle, including mechanisms of spindle assembly. Comparison with extracts from the diploid Western clawed frog, Xenopus tropicalis, which is smaller at the organism, cellular and subcellular levels, has enabled the identification of spindle size scaling factors. We set out to characterize the Marsabit clawed frog, Xenopus borealis, which is intermediate in size between the two species, but more recently diverged in evolution from X. laevis than X. tropicalis. X. borealis eggs were slightly smaller than those of X. laevis, and slightly smaller spindles were assembled in egg extracts. Interestingly, microtubule distribution across the length of the X. borealis spindles differed from both X. laevis and X. tropicalis. Extract mixing experiments revealed common scaling phenomena among Xenopus species, while characterization of spindle factors katanin, TPX2, and Ran indicate that X. borealis spindles possess both X. laevis and X. tropicalis features. Thus, X. borealis egg extract provides a third in vitro system to investigate interspecies scaling and spindle morphometric variation.

Asunto(s)

Óvulo , Huso Acromático , Xenopus , Animales , Extractos Celulares , Femenino

RESUMEN

Nuclear pore complexes (NPCs) are gateways through the nuclear envelope. How they form into a structure containing three rings and integrate into the nuclear envelope remains a challenging paradigm for coordinated assembly of macro-complexes. In vertebrates, the cytoplasmic and nucleoplasmic rings of NPCs are mostly formed by multiple copies of the Nup107-Nup160 complex, whereas the central, or inner ring is composed of Nup53, Nup93, Nup155 and the two paralogues Nup188 and Nup205. Inner ring assembly is only partially understood. Using in vitro nuclear assembly reactions, we show that direct pore membrane binding of Nup155 is crucial for NPC formation. Replacing full-length Nup155 with its N-terminal ß-propeller allows assembly of the outer ring components to the NPC backbone that also contains Nup53. However, further assembly, especially recruitment of the Nup93 and Nup62 complexes, is blocked. Self-interaction between the N- and C-terminal domains of Nup155 has an auto-inhibitory function that prevents interaction between the N-terminus of Nup155 and the C-terminal region of Nup53. Nup93 can overcome this block by binding to Nup53, thereby promoting formation of the inner ring and the NPC.

Asunto(s)

Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Sitios de Unión , Proteínas de Complejo Poro Nuclear/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Xenopus/genética , Xenopus laevis

RESUMEN

The minichromosome maintenance (MCM) complex, consisting of six subunits, Mcm2-7, is loaded onto replication origins through loading factors (origin recognition complex [ORC], Cdc6, and Cdt1) and forms an MCM double hexamer that licenses the initiation of DNA replication. Previous studies with Xenopus egg extracts showed that loading factors, especially Cdc6, dissociate from chromatin on MCM loading, but the molecular mechanism and physiological significance remain largely unknown. Using a cell-free system for MCM loading onto plasmid DNA in Xenopus egg extracts, we found that MCM loaded onto DNA prevents DNA binding of the loading factors ORC, Cdc6, and Cdt1. We further report that a peptide of the C-terminal region of MCM3 (MCM3-C), previously implicated in the initial association with ORC/Cdc6 in budding yeast, prevents ORC/Cdc6/Cdt1 binding to DNA in the absence of MCM loading. ATP-γ-S suppresses inhibitory activities of both the MCM loaded onto DNA and the MCM3-C peptide. Other soluble factors in the extract, but neither MCM nor Cdt1, are required for the activity. Conservation of the amino acid sequences of MCM3-C and its activity in vertebrates implies a novel negative autoregulatory mechanism that interferes with MCM loading in the vicinity of licensed origins to ensure proper origin licensing.

Asunto(s)

Replicación del ADN , Componente 3 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Animales , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , ADN/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Componente 3 del Complejo de Mantenimiento de Minicromosoma/química , Componente 3 del Complejo de Mantenimiento de Minicromosoma/genética , Complejo de Reconocimiento del Origen/química , Complejo de Reconocimiento del Origen/metabolismo , Óvulo/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Dominios Proteicos , Alineación de Secuencia , Proteínas de Xenopus/química , Proteínas de Xenopus/genética

RESUMEN

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.

Asunto(s)

Proteínas de Ciclo Celular/genética , Cromatina/química , Replicación del ADN , Proteínas de Unión al ADN/genética , Geminina/genética , Óvulo/química , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Sitios de Unión , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Cromatina/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Geminina/metabolismo , Expresión Génica , Masculino , Mutación , Óvulo/citología , Óvulo/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espermatozoides/química , Espermatozoides/citología , Espermatozoides/metabolismo , Relación Estructura-Actividad , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo

RESUMEN

Cell-free extracts made from Xenopus laevis eggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion. A powerful approach involving immunodepletion of cohesin and its regulators is also included.

Asunto(s)

Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Biología Molecular/métodos , Intercambio de Cromátides Hermanas/genética , Animales , Cromátides/genética , Cromatina/genética , Replicación del ADN/genética , Mitosis/genética , Óvulo/crecimiento & desarrollo , Óvulo/metabolismo , Extractos de Tejidos/genética , Xenopus laevis/genética , Cohesinas

RESUMEN

Cdt1 is a protein essential for initiation of DNA replication; it recruits MCM helicase, a core component of the replicative DNA helicase, onto replication origins. In our previous study, we showed that addition of excess Cdt1 inhibits nascent strand elongation during DNA replication in Xenopus egg extracts. In the present study, we investigated the mechanism behind the inhibitory effect of Cdt1. We found that addition of recombinant Cdt1 inhibited nascent DNA synthesis in a reinitiation-independent manner. To identify the mechanism by which Cdt1 inhibits nascent strand elongation, the effect of Cdt1 on loading of Mcm4 and Rpa70 onto chromatin was examined. The results showed that Cdt1 suppressed the excessive Rpa70 binding caused by extensive, aphidicolin-induced DNA unwinding; this unwinding occurs between stalled DNA polymerases and advancing replication forks. These findings suggested that excess Cdt1 suppressed the progression of replication forks.

Asunto(s)

Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Óvulo/fisiología , Elongación de la Transcripción Genética/fisiología , Animales , Xenopus laevis

RESUMEN

The cytotoxicity of SN1-type alkylating agents such as N-methyl-N'-nitrosourea (MNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), or the cancer chemotherapeutics temozolomide, dacarbazine and streptozotocin has been ascribed to the persistence of O(6)-methylguanine ((me)G) in genomic DNA. One hypothesis posits that (me)G toxicity is caused by futile attempts of the mismatch repair (MMR) system to process (me)G/C or (me)G/T mispairs arising during replication, while an alternative proposal suggests that the latter lesions activate DNA damage signaling, cell cycle arrest and apoptosis directly. Attempts to elucidate the molecular mechanism of (me)G-induced cell killing in vivo have been hampered by the fact that the above reagents induce several types of modifications in genomic DNA, which are processed by different repair pathways. In contrast, defined substrates studied in vitro did not undergo replication. We set out to re-examine this phenomenon in replication-competent Xenopus laevis egg extracts, using either phagemid substrates containing a single (me)G residue, or methylated sperm chromatin. Our findings provide further support for the futile cycling hypothesis.

Asunto(s)

Daño del ADN , Reparación de la Incompatibilidad de ADN/fisiología , ADN/metabolismo , Guanina/análogos & derivados , Animales , Extractos Celulares , ADN/química , Replicación del ADN , Guanina/metabolismo , Óvulo/metabolismo , Xenopus laevis

RESUMEN

Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase α and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.

Asunto(s)

Replicación del ADN , RecQ Helicasas/fisiología , Origen de Réplica , Proteínas de Xenopus/fisiología , Animales , Sitios de Unión , Extractos Celulares , Sistema Libre de Células , Células Cultivadas , Oocitos , Plásmidos/genética , Factores de Transcripción/fisiología , Xenopus laevis