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Shusuke Tada

Publications and source records attributed to Shusuke Tada.

16 recordsLinked to original sources
PubMed2007

Cdt1 and geminin: role during cell cycle progression and DNA damage in higher eukaryotes.

DNA replication in eukaryotic cells must be strictly regulated to ensure that the entire genome is duplicated only once in each cell cycle. For this purpose, the initiation of DNA replication is controlled by the "licensing" reaction, which is established by the formation of a pre-replicative complex (pre-RC) with the sequential assembly of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 onto origin regions. Among these, Cdt1 is likely the most important target for regulating licensing in higher eukaryotic cells, since illegitimate accumulation of Cdt1 causes multiple rounds of DNA replication without an intervening mitosis. Cdt1 is regulated over the course of the cell cycle mainly by the controlled expression of an inhibitor protein, geminin, and the level of Cdt1 periodically fluctuates due to ubiquitination and proteolysis. While the expression of geminin from S phase to metaphase of mitosis prevents licensing, Cdt1 accumulates from M to G1 phases and is degraded at the onset of S phase. Furthermore, Cdt1 is also proteolyzed in G1 phase in response to DNA damage, presumably providing a new checkpoint control.

Animals↗
PubMed2006

Chromatin loading of Smc5/6 is induced by DNA replication but not by DNA double-strand breaks.

Smc6, a member of the structural maintenance of chromosomes (SMC) family of proteins, forms a complex with related Smc5. Genetic analyses of yeast have demonstrated the involvement of Smc6 in DNA repair and checkpoint responses. In this study, we investigated the role of the Smc5/6 complex in higher eukaryotes by analyzing its behavior in Xenopus laevis egg extracts. Smc5/6 was loaded onto chromatin during DNA replication in a manner dependent on the initiation of DNA synthesis, and it dissociated from chromatin during mitosis. Moreover, the induction of DNA double-strand breaks following replication did not significantly affect the amount of chromatin-associated Smc6. These findings suggest that the Smc5/6 complex is regulated during the cell cycle, presumably in anticipation of DNA damage that may arise during replication.

Animals↗
PubMed2006

A novel Rad18 function involved in protection of the vertebrate genome after exposure to camptothecin.

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.

Animals↗
PubMed2006

BLM is an early responder to DNA double-strand breaks.

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a marked predisposition to cancer and elevated genomic instability. The defective protein in BS, BLM, is a member of the RecQ helicase family and is believed to function in various DNA transactions, including in replication, repair, and recombination. Here, we show that both endogenous and overexpressed human BLM accumulates at sites of laser light-induced DNA double-strand breaks within 10s and colocalizes with gammaH2AX and ATM. Like its RecQ helicase family member, WRN, the defective protein in Werner syndrome, dissection of the BLM protein revealed that its HRDC domain is sufficient for its recruitment to the damaged sites. In addition, we confirmed that the C-terminal region spanning amino acids 1250-1292 within the HRDC domain is necessary for BLM recruitment. To identify additional proteins required for the recruitment of BLM, we examined the recruitment of BLM in various mutants generated from chicken DT40 cells and found that the early accumulation of BLM was not dependent on the presence of ATM, RAD17, DNA-PKcs, NBS1, XRCC3, RAD52, RAD54, or WRN. Thus, HRDC domain in DNA helicases is a common early responder to DNA double-strand breaks, enabling BLM and WRN to be involved in DNA repair.

Adenosine Triphosphatases↗
PubMed2006

Analyses of the interaction of WRNIP1 with Werner syndrome protein (WRN) in vitro and in the cell.

Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2006

Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair.

Dpb11 is required for the loading of DNA polymerases alpha and epsilon on to DNA in chromosomal DNA replication and interacts with the DNA damage checkpoint protein Ddc1 in Saccharomyces cerevisiae. The interaction between the homologs of Dpb11 and Ddc1 in human cells and fission yeast is thought to reflect their involvement in the checkpoint response. Here we show that dpb11-1 cells, carrying a mutated Dpb11 that cannot interact with Ddc1, are defective in the repair of methyl methanesulfonate (MMS)-induced DNA damage but not in the DNA damage checkpoint at the permissive temperature. Epistatic analyses suggested that Dpb11 is involved in the Rad51/Rad52-dependent recombination pathway. Ddc1 as well as Dpb11 were required for homologous recombination induced by MMS. Moreover, we found the in vivo association of Dpb11 and Ddc1 with not only the HO-induced double-strand break (DSB) site at MAT locus but also the donor sequence HML during homologous recombination between MAT and HML. Rad51 was required for their association with the HML donor locus, but not with DSB site at the MAT locus. In addition, the association of Dpb11 with the MAT and HML locus after induction of HO-induced DSB was dependent on Ddc1. These results indicate that, besides the involvement in the replication and checkpoint, Dpb11 functions with Ddc1 in the recombination repair process itself.

Cell Cycle Proteins↗
PubMed2006

Bloom helicase and DNA topoisomerase IIIalpha are involved in the dissolution of sister chromatids.

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.

2-Aminopurine↗
PubMed2006

Functional relationships between Rad18 and WRNIP1 in vertebrate cells.

The WRNIP1 protein interacts with WRN, the product of the causative gene for Werner syndrome. Mutation of the Saccharomyces cerevisiae gene MGS1, the yeast counterpart of WRNIP1, confers synthetic lethality with mutation of RAD18. To examine the functional relationship between WRNIP1 and Rad18 in higher eukaryotic cells, we generated WRNIP1-/-/-/RAD18-/- lines from chicken DT40 cells and compared them with single mutant cell lines. Unlike the corresponding yeast mutant, WRNIP1-/-/-/RAD18-/- cells are viable but grow more slowly than single mutants and wild type cells, and they show an additive or synergistic elevation in the frequency of sister chromatid exchanges. As reported, WRNIP1-/-/- cells and RAD18-/- cells are moderately and severely sensitive to camptothecin (CPT), respectively. Unexpectedly, the severe CPT sensitivity of RAD18-/- cells is slightly suppressed by disruption of the WRNIP1 gene.

Animals↗
PubMed2005

Caenorhabditis elegans geminin homologue participates in cell cycle regulation and germ line development.

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins.

Amino Acid Sequence↗
PubMed2005

Licensing for DNA replication requires a strict sequential assembly of Cdc6 and Cdt1 onto chromatin in Xenopus egg extracts.

Replication origins are licensed for a single initiation event by the loading of Mcm2-7 proteins during late mitosis and G1. Sequential associations of origin recognition complex, Cdc6 and Mcm2-7 are essential for completion of the licensing. Although Cdt1 also binds to the chromatin when the licensing reaction takes place, whether the binding is a requirement for Cdt1 to function is unclear. To analyze the relevance of the chromatin association of Cdt1, we carried out chromatin transfer experiments using either immunodepleted Xenopus egg extracts or purified proteins. Licensing assay and immunoblotting analyses indicated that Cdt1 could only license DNA replication and load Mcm2-7 onto DNA when it binds to chromatin that has already associated with Cdc6. These results provide evidence supporting that Cdc6 and Cdt1 must bind to chromatin in a strict order for DNA licensing to occur.

Animals↗
PubMed2004

Structural basis for inhibition of the replication licensing factor Cdt1 by geminin.

To maintain chromosome stability in eukaryotic cells, replication origins must be licensed by loading mini-chromosome maintenance (MCM2-7) complexes once and only once per cell cycle. This licensing control is achieved through the activities of geminin and cyclin-dependent kinases. Geminin binds tightly to Cdt1, an essential component of the replication licensing system, and prevents the inappropriate reinitiation of replication on an already fired origin. The inhibitory effect of geminin is thought to prevent the interaction between Cdt1 and the MCM helicase. Here we describe the crystal structure of the mouse geminin-Cdt1 complex using tGeminin (residues 79-157, truncated geminin) and tCdt1 (residues 172-368, truncated Cdt1). The amino-terminal region of a coiled-coil dimer of tGeminin interacts with both N-terminal and carboxy-terminal parts of tCdt1. The primary interface relies on the steric complementarity between the tGeminin dimer and the hydrophobic face of the two short N-terminal helices of tCdt1 and, in particular, Pro 181, Ala 182, Tyr 183, Phe 186 and Leu 189. The crystal structure, in conjunction with our biochemical data, indicates that the N-terminal region of tGeminin might be required to anchor tCdt1, and the C-terminal region of tGeminin prevents access of the MCM complex to tCdt1 through steric hindrance.

Animals↗
PubMed2004

The absence of a functional relationship between ATM and BLM, the components of BASC, in DT40 cells.

Bloom syndrome (BS) and ataxia-telangiectasia (A-T) are rare autosomal recessive diseases associated with chromosomal instability. The genes responsible for BS and A-T have been identified as BLM and ATM, respectively, whose products were recently found to be components of BRCA1-associated genome surveillance complex (BASC), a supercomplex possibly involved in the recognition and repair of aberrant DNA structures. Based on experiments using BLM(-/-) DT40 cells and BLM(-/-)/RAD54(-/-) DT40 cells, we previously suggested that BLM functions to reduce the formation of double-strand breaks (DSBs) during DNA replication. To examine whether ATM is involved in the recognition and/or repair of DSBs generated in BLM(-/-) DT40 cells and to address the functional relationship between the two BASC components, we generated BLM(-/-)/ATM(-/-) DT40 cells and characterized their properties as well as those of ATM(-/-) and BLM(-/-) DT40 cells. BLM(-/-)/ATM(-/-) cells proliferated slightly more slowly than either BLM(-/-) or ATM(-/-) cells. The sensitivity of BLM(-/-)/ATM(-/-) cells to gamma-irradiation was similar to that of ATM(-/-) cells, while BLM(-/-) cells were slightly resistant to gamma-irradiation compared with wild-type cells. BLM(-/-) cells showed sensitivity to methyl methanesulfonate (MMS) and UV irradiation while ATM(-/-) cells did not show sensitivity to either agent. The sensitivity of BLM(-/-)/ATM(-/-) cells to MMS and UV was similar to that of BLM(-/-) cells. Disrupting the function of ATM reduced the targeted integration frequency in BLM(-/-) DT40 cells. However, a defect in ATM only slightly reduced the increased sister chromatid exchanges (SCEs) in BLM(-/-) DT40 cells.

Adenosine Triphosphatases↗
PubMed2003

Functional relation among RecQ family helicases RecQL1, RecQL5, and BLM in cell growth and sister chromatid exchange formation.

Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.

Adenosine Triphosphatases↗
PubMed2002

Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract.

The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.

Animals↗
PubMed2002

Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import.

During late mitosis and early interphase, origins of replication become "licensed" for DNA replication by loading Mcm2-7 complexes. Mcm2-7 complexes are removed from origins as replication forks initiate replication, thus preventing rereplication of DNA in a single cell cycle. Premature origin licensing is prevented in metaphase by the action of geminin, which binds and inhibits Cdt1/RLF-B, a protein that is required for the loading of Mcm2-7. Recombinant geminin that is added to Xenopus egg extracts is efficiently degraded upon exit from metaphase. Here, we show that recombinant and endogenous forms of Xenopus geminin behave differently from one another, such that a significant proportion of endogenous geminin escapes proteolysis upon exit from metaphase. During late mitosis and early G1, the surviving population of endogenous geminin does not associate with Cdt1/RLF-B and does not inhibit licensing. Following nuclear assembly, geminin is imported into nuclei and becomes reactivated to bind Cdt1/RLF-B. This reactivated geminin provides the major nucleoplasmic inhibitor of origin relicensing during late interphase. Since the initiation of replication at licensed origins depends on nuclear assembly, our results suggest an elegant and novel mechanism for preventing rereplication of DNA in a single cell cycle.

Active Transport, Cell Nucleus↗