Publications by authors named "Shusuke Tada"

52 Publications

N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.

Gene 2021 Jun 15;787:145647. Epub 2021 Apr 15.

Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan. Electronic address:

RecQ4, a member of the RecQ helicase family, is required for the maintenance of genome integrity. RecQ4 has been shown to promote the following two DNA double-strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). However, its molecular function has not been fully elucidated. In the present study, we aimed to investigate the role of RecQ4 in NHEJ using Xenopus egg extracts. The N-terminal 598 amino acid region of Xenopus RecQ4 (N598), which lacks a central helicase domain and a downstream C-terminal region, was added to the extracts and its effect on the joining of DNA ends was analyzed. We found that N598 inhibited the joining of linearized DNA ends in the extracts. In addition, N598 inhibited DSB-induced chromatin binding of Ku70, which is essential for NHEJ, while the DSB-induced chromatin binding of the HR-associated proteins, replication protein A (RPA) and Rad51, increased upon the addition of N598. These results suggest that RecQ4 possibly influences the choice of the DSB repair pathway by influencing the association of the Ku heterodimer with the DNA ends.
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http://dx.doi.org/10.1016/j.gene.2021.145647DOI Listing
June 2021

Biphasic Increases of Cell Surface Calreticulin Following Treatment with Mitoxantrone.

Biol Pharm Bull 2020 Oct 29;43(10):1595-1599. Epub 2020 Jul 29.

Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University.

Calreticulin (CRT) and calnexin (CNX), homologous major chaperones in the endoplasmic reticulum (ER), are known to translocate to the cell surface in response to chemotherapeutic agents, such as mitoxantrone (MIT), and cellular stresses, including apoptosis. Cell surface CRT (ecto-CRT) is relevant to the phagocytic uptake of cancer cells and dying cells, and pre-apoptotic exposure of CRT has been reported to result in enhanced immunogenicity of dying tumor cells, serving as a damage-associated molecular pattern (DAMP). In this study, HT-29 cells were treated with MIT to induce ER stress, and ecto-CRT and cell surface CNX were quantified by flow cytometry in the absence or presence of caspase inhibitors, a calpain inhibitor, or a scavenger of reactive oxygen species. The biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells was observed after treatment with MIT. We confirmed that the early increase in ecto-CRT after 4 h of MIT treatment was not related to apoptosis, whereas the increase of ecto-CRT, as well as that of cell-surface CNX, during the later stage of treatment was caspase dependent and related to apoptosis. In addition, our results suggested that the early peak of ecto-CRT was mediated by activation of caspase 8 by ER stress. Thus, the physiological significance of the late increases in cell-surface CRT and/or CNX might be considered an "eat-me signal" for the removal of dead cells by phagocytosis, while the early increase in ecto-CRT caused by ER stress might enhance the immunogenicity of stressed tumor cells.
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http://dx.doi.org/10.1248/bpb.b20-00319DOI Listing
October 2020

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

Biochem Biophys Res Commun 2017 09 8;490(4):1375-1380. Epub 2017 Jul 8.

Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba 274-8510, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.034DOI Listing
September 2017

Excess Cdt1 inhibits nascent strand elongation by repressing the progression of replication forks in Xenopus egg extracts.

Biochem Biophys Res Commun 2016 Feb 8;470(2):405-410. Epub 2016 Jan 8.

Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Nakano-ku, Tokyo, Japan; Department of Molecular Biology, Faculty of Pharmaceutical Sciences, Toho University, Funabashi-shi, Chiba, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbrc.2016.01.028DOI Listing
February 2016

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response.

Biochem Biophys Res Commun 2014 Sep 16;452(1):48-52. Epub 2014 Aug 16.

Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH(-/-)) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.
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http://dx.doi.org/10.1016/j.bbrc.2014.08.043DOI Listing
September 2014

Tipin functions in the protection against topoisomerase I inhibitor.

J Biol Chem 2014 Apr 25;289(16):11374-11384. Epub 2014 Feb 25.

Department of Biochemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan,. Electronic address:

The replication fork temporarily stalls when encountering an obstacle on the DNA, and replication resumes after the barrier is removed. Simultaneously, activation of the replication checkpoint delays the progression of S phase and inhibits late origin firing. Camptothecin (CPT), a topoisomerase I (Top1) inhibitor, acts as a DNA replication barrier by inducing the covalent retention of Top1 on DNA. The Timeless-Tipin complex, a component of the replication fork machinery, plays a role in replication checkpoint activation and stabilization of the replication fork. However, the role of the Timeless-Tipin complex in overcoming the CPT-induced replication block remains elusive. Here, we generated viable TIPIN gene knock-out (KO) DT40 cells showing delayed S phase progression and increased cell death. TIPIN KO cells were hypersensitive to CPT. However, homologous recombination and replication checkpoint were activated normally, whereas DNA synthesis activity was markedly decreased in CPT-treated TIPIN KO cells. Proteasome-dependent degradation of chromatin-bound Top1 was induced in TIPIN KO cells upon CPT treatment, and pretreatment with aphidicolin, a DNA polymerase inhibitor, suppressed both CPT sensitivity and Top1 degradation. Taken together, our data indicate that replication forks formed without Tipin may collide at a high rate with Top1 retained on DNA by CPT treatment, leading to CPT hypersensitivity and Top1 degradation in TIPIN KO cells.
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http://dx.doi.org/10.1074/jbc.M113.531707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036274PMC
April 2014

Rmi1 functions in S phase-mediated cohesion establishment via a pathway involving the Ctf18-RFC complex and Mrc1.

Biochem Biophys Res Commun 2012 Oct 1;427(3):682-6. Epub 2012 Oct 1.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

Saccharomyces cerevisiae RecQ helicase (Sgs1) combines with DNA topoisomerase III (Top3) and RecQ-mediated genome instability 1 (Rmi1) to form an evolutionarily conserved complex that is required for processing homologous recombination intermediates and restarting collapsed replication forks. It was previously reported that Rmi1 contributes to sister chromatid cohesion; however, the underlying molecular mechanism has been unclear. In the present study, Rmi1 was found to be enriched at the region close to an early-firing replication origin when replication forks were arrested near their origins in the presence of hydroxyurea. Genetic analyses revealed that Rmi1 promoted sister chromatid cohesion in a process that was distinct from both the cohesion establishment pathway involving Ctf4, Csm3, and Chl1 and the pathway involving the acetylation of Smc3. On the other hand, Rmi1 seemed to function in a pathway involving the Ctf18-RFC complex and Mrc1, which were previously predicted to regulate leading-strand DNA replication.
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http://dx.doi.org/10.1016/j.bbrc.2012.09.124DOI Listing
October 2012

Functional relationship between Claspin and Rad17.

Biochem Biophys Res Commun 2011 Oct 14;414(2):298-303. Epub 2011 Sep 14.

Molecular Cell Biology Laboratory, Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, 1-1-20 Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan.

Claspin was originally identified as a Check1 (Chk1)-interacting protein. Claspin and Rad17 are reportedly involved in the DNA damage-induced phosphorylation of Chk1, a hallmark of checkpoint activation. To understand the cellular functions of Claspin and the functional relationship between Claspin and Rad17, we generated Claspin(-/-) and Claspin(-/-)/RAD17(-) cells using chicken DT40 cells, which contain an exogenously introduced Claspin that can be suppressed by the addition of doxycycline (Dox). In the presence of Dox, Claspin(-/-) cells ceased growth within 2 days, leading to cell death. In addition, a remarkable reduction in the rate of DNA elongation was observed in Claspin-depleted cells, suggesting that Claspin plays a critical role in DNA replication in the absence of exogenous stress. When cells were exposed to methyl methanesulfonate (MMS), a DNA damaging agent, RAD17(-) cells showed a greater defect in checkpoint activation than Claspin(-/-) cells as monitored by progression of cell cycle and phosphorylation of Chk1. Knocking out RAD17 gene showed almost no additive effects on cell death and DNA elongation rates in Claspin-depleted cells.
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http://dx.doi.org/10.1016/j.bbrc.2011.09.037DOI Listing
October 2011

Roles of histone chaperone CIA/Asf1 in nascent DNA elongation during nucleosome replication.

Genes Cells 2011 Oct 7;16(10):1050-62. Epub 2011 Sep 7.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi, Japan.

The nucleosome, which is composed of DNA wrapped around a histone octamer, is a fundamental unit of chromatin and is duplicated during the eukaryotic DNA replication process. The evolutionarily conserved histone chaperone cell cycle gene 1 (CCG1) interacting factor A/anti-silencing function 1 (CIA/Asf1) is involved in histone transfer and nucleosome reassembly during DNA replication. CIA/Asf1 has been reported to split the histone (H3-H4)(2) tetramer into histone H3-H4 dimer(s) in vitro, raising a possibility that, in DNA replication, CIA/Asf1 is involved in nucleosome disassembly and the promotion of semi-conservative histone H3-H4 dimer deposition onto each daughter strand in vivo. Despite numerous studies on the functional roles of CIA/Asf1, its mechanistic role(s) remains elusive because of lack of biochemical analyses. The biochemical studies described here show that a V94R CIA/Asf1 mutant, which lacks histone (H3-H4)(2) tetramer splitting activity, does not form efficiently a quaternary complex with histones H3-H4 and the minichromosome maintenance 2 (Mcm2) subunit of the Mcm2-7 replicative DNA helicase. Interestingly, the mutant enhances nascent DNA strand synthesis in a cell-free chromosomal DNA replication system using Xenopus egg extracts. These results suggest that CIA/Asf1 in the CIA/Asf1-H3-H4-Mcm2 complex, which is considered to be an intermediate in histone transfer during DNA replication, negatively regulates the progression of the replication fork.
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http://dx.doi.org/10.1111/j.1365-2443.2011.01549.xDOI Listing
October 2011

Werner interacting protein 1 promotes binding of Werner protein to template-primer DNA.

Biol Pharm Bull 2011 ;34(8):1314-8

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980–8578, Japan.

Werner interacting protein 1 (WRNIP1) that is highly conserved from Escherichia coli to human was originally identified as a protein that interacts with the Werner syndrome responsible gene product (WRN). Here, human WRNIP1 and WRN are shown to bind to template-primer DNA, and WRNIP1, but not WRN, requires ATP for DNA binding. Under conditions of a limiting amount of WRN, WRNIP1 facilitated binding of WRN to DNA in a dose-dependent manner. However, WRNIP1 did not stimulate the DNA helicase activity of WRN, and WRN displaced pre-bound WRNIP1 from DNA. Functional relationships between WRNIP1 and WRN will be discussed.
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http://dx.doi.org/10.1248/bpb.34.1314DOI Listing
February 2012

The histone chaperone facilitates chromatin transcription (FACT) protein maintains normal replication fork rates.

J Biol Chem 2011 Sep 7;286(35):30504-30512. Epub 2011 Jul 7.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578; Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo 202-8585. Electronic address:

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.
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http://dx.doi.org/10.1074/jbc.M111.264721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3162410PMC
September 2011

The N-terminal region of RECQL4 lacking the helicase domain is both essential and sufficient for the viability of vertebrate cells. Role of the N-terminal region of RECQL4 in cells.

Biochim Biophys Acta 2011 Mar 20;1813(3):473-9. Epub 2011 Jan 20.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.
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http://dx.doi.org/10.1016/j.bbamcr.2011.01.001DOI Listing
March 2011

Biphasic chromatin binding of histone chaperone FACT during eukaryotic chromatin DNA replication.

Biochim Biophys Acta 2011 Jun 11;1813(6):1129-36. Epub 2011 Jan 11.

Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

The facilitates chromatin transcription (FACT) complex affects nuclear DNA transactions in a chromatin context. Though the involvement of FACT in eukaryotic DNA replication has been revealed, a clear understanding of its biochemical behavior during DNA replication still remains elusive. Here, we analyzed the chromatin-binding dynamics of FACT using Xenopus egg extract cell-free system. We found that FACT has at least two distinct chromatin-binding phases: (1) a rapid chromatin-binding phase at the onset of DNA replication that did not involve origin licensing and (2) a second phase of chromatin binding that initiated after origin licensing. Intriguingly, early-binding FACT dissociated from chromatin when DNA replication was blocked by the addition of Cdc6 in the licensed state before origin firing. Cdc6-induced removal of FACT was blocked by the inhibition of origin licensing with geminin, but not by suppressing the activity of DNA polymerases, CDK, or Cdc7. Furthermore, chromatin transfer experiments revealed that impairing the later binding of FACT severely compromises DNA replication activity. Taken together, we propose that even though FACT has rapid chromatin-binding activity, the binding pattern of FACT on chromatin changes after origin licensing, which may contribute to the establishment of its functional link to the DNA replication machinery.
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http://dx.doi.org/10.1016/j.bbamcr.2011.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428913PMC
June 2011

SOD1 Is Essential for the Viability of DT40 Cells and Nuclear SOD1 Functions as a Guardian of Genomic DNA.

J Nucleic Acids 2010 Aug 5;2010. Epub 2010 Aug 5.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

Reactive oxygen species (ROSs) are produced during normal cellular metabolism, particularly by respiration in mitochondria, and these ROSs are considered to cause oxidative damage to macromolecules, including DNA. In our previous paper, we found no indication that depletion of mitochondrial superoxide dismutase, SOD2, resulted in an increase in DNA damage. In this paper, we examined SOD1, which is distributed in the cytoplasm, nucleus, and mitochondrial intermembrane space. We generated conditional SOD1 knockout cells from chicken DT40 cells and analyzed their phenotypes. The results revealed that SOD1 was essential for viability and that depletion of SOD1, especially nuclear SOD1, increased sister chromatid exchange (SCE) frequency, suggesting that superoxide is generated in or near the nucleus and that nuclear SOD1 functions as a guardian of the genome. Furthermore, we found that ascorbic acid could offset the defects caused by SOD1 depletion, including cell lethality and increases in SCE frequency and apurinic/apyrimidinic sites.
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http://dx.doi.org/10.4061/2010/795946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2929635PMC
August 2010

Deregulated Cdc6 inhibits DNA replication and suppresses Cdc7-mediated phosphorylation of Mcm2-7 complex.

Nucleic Acids Res 2010 Sep 26;38(16):5409-18. Epub 2010 Apr 26.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

Mcm2-7 is recruited to eukaryotic origins of DNA replication by origin recognition complex, Cdc6 and Cdt1 thereby licensing the origins. Cdc6 is essential for origin licensing during DNA replication and is readily destabilized from chromatin after Mcm2-7 loading. Here, we show that after origin licensing, deregulation of Cdc6 suppresses DNA replication in Xenopus egg extracts without the involvement of ATM/ATR-dependent checkpoint pathways. DNA replication is arrested specifically after chromatin binding of Cdc7, but before Cdk2-dependent pathways and deregulating Cdc6 after this step does not impair activation of origin firing or elongation. Detailed analyses revealed that Cdc6 deregulation leads to strong suppression of Cdc7-mediated hyperphosphorylation of Mcm4 and subsequent chromatin loading of Cdc45, Sld5 and DNA polymerase α. Mcm2 phosphorylation is also repressed although to a lesser extent. Remarkably, Cdc6 itself does not directly inhibit Cdc7 kinase activity towards Mcm2-4-6-7 in purified systems, rather modulates Mcm2-7 phosphorylation on chromatin context. Taken together, we propose that Cdc6 on chromatin acts as a modulator of Cdc7-mediated phosphorylation of Mcm2-7, and thus destabilization of Cdc6 from chromatin after licensing is a key event ensuring proper transition to the initiation of DNA replication.
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http://dx.doi.org/10.1093/nar/gkq262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2938227PMC
September 2010

Structure of the Cdt1 C-terminal domain: conservation of the winged helix fold in replication licensing factors.

Protein Sci 2009 Nov;18(11):2252-64

The Magnetic Resonance Team, Korea Basic Science Institute, 804-1 Yangchung-Ri, Ochang, Chungbuk 363-883, South Korea.

In eukaryotic replication licensing, Cdt1 plays a key role by recruiting the MCM2-7 complex onto the origin of chromosome. The C-terminal domain of mouse Cdt1 (mCdt1C), the most conserved region in Cdt1, is essential for licensing and directly interacts with the MCM2-7 complex. We have determined the structures of mCdt1CS (mCdt1C_small; residues 452 to 557) and mCdt1CL (mCdt1C_large; residues 420 to 557) using X-ray crystallography and solution NMR spectroscopy, respectively. While the N-terminal 31 residues of mCdt1CL form a flexible loop with a short helix near the middle, the rest of mCdt1C folds into a winged helix structure. Together with the middle domain of mouse Cdt1 (mCdt1M, residues 172-368), this study reveals that Cdt1 is formed with a tandem repeat of the winged helix domain. The winged helix fold is also conserved in other licensing factors including archaeal ORC and Cdc6, which supports an idea that these replication initiators may have evolved from a common ancestor. Based on the structure of mCdt1C, in conjunction with the biochemical analysis, we propose a binding site for the MCM complex within the mCdt1C.
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http://dx.doi.org/10.1002/pro.236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2788280PMC
November 2009

Physical and functional interaction between WRNIP1 and RAD18.

Genes Genet Syst 2009 Apr;84(2):171-8

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

WRN interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product (WRN). WRNIP1 is a highly conserved protein from E. coli to humans. Genetic studies in budding yeast suggested that the yeast orthlog of WRNIP1, Mgs1, may function in a DNA damage tolerance pathway that is similar to, but distinct from, the template-switch damage avoidance pathway involving Rad6, Rad18, Rad5, Mms2, and Ubc13. Here we report that human WRNIP1 binds in an ATP dependent manner to both forked DNA that mimics stalled replication forks and to template/primer DNA. We found that WRNIP1 interacts physically with RAD18 and interferes with the binding of RAD18 to forked DNA and to template/primer DNA. In contrast, RAD18 enhances the binding of WRNIP1 to these DNAs, suggesting that WRNIP1 targets DNA bound by RAD18.
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http://dx.doi.org/10.1266/ggs.84.171DOI Listing
April 2009

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication.

DNA Repair (Amst) 2009 Jun 3;8(6):690-6. Epub 2009 Mar 3.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA-damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, chromatin immunoprecipitation (ChIP) on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double mutation of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD53 single mutation. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed.
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http://dx.doi.org/10.1016/j.dnarep.2009.01.018DOI Listing
June 2009

A novel role for Rad17 in homologous recombination.

Genes Genet Syst 2008 Oct;83(5):427-31

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Replication checkpoint protein Rad17 senses DNA lesions during DNA replication and halts progression of replication fork. The cells derived from Bloom syndrome individuals show some defects in DNA replication. In order to investigate the functional relationship between the replication checkpoint protein Rad17 and BLM, which is the product of the causative gene of Bloom syndrome, we generated BLM/RAD17 double knockout (blm/rad17) cells using chicken DT40 cells. The blm/rad17 cells showed exaggerated growth defects as determined by analysis of their growth curves and plating efficiency compared to those of either of the single gene mutants. These defects seem to be due to an increase in DNA lesions that cause spontaneous cell death, suggesting that Rad17 and BLM execute different functions in the progression of replication forks. We also demonstrate that targeting integration was dramatically compromised by a lack of Rad17. In addition, the elevated frequency of sister chromatid exchange (SCE) due to homologous recombination in BLM knockout (blm) cells was greatly reduced by disruption of the RAD17 gene. Thus, in addition to its role in the replication checkpoint, Rad17 appears to play a role in homologous recombination.
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http://dx.doi.org/10.1266/ggs.83.427DOI Listing
October 2008

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2.

Biochem Biophys Res Commun 2009 Feb 25;379(2):233-8. Epub 2008 Dec 25.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.
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http://dx.doi.org/10.1016/j.bbrc.2008.12.031DOI Listing
February 2009

Repression of nascent strand elongation by deregulated Cdt1 during DNA replication in Xenopus egg extracts.

Mol Biol Cell 2009 Feb 8;20(3):937-47. Epub 2008 Dec 8.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, 980-8578, Japan.

Excess Cdt1 reportedly induces rereplication of chromatin in cultured cells and Xenopus egg extracts, suggesting that the regulation of Cdt1 activity by cell cycle-dependent proteolysis and expression of the Cdt1 inhibitor geminin is crucial for the inhibition of chromosomal overreplication between S phase and metaphase. We analyzed the consequences of excess Cdt1 for DNA replication and found that increased Cdt1 activity inhibited the elongation of nascent strands in Xenopus egg extracts. In Cdt1-supplemented extracts, overreplication was remarkably induced by the further addition of the Cdt1-binding domain of geminin (Gem79-130), which lacks licensing inhibitor activity. Further analyses indicated that fully active geminin, as well as Gem79-130, restored nascent strand elongation in Cdt1-supplemented extracts even after the Cdt1-induced stalling of replication fork elongation had been established. Our results demonstrate an unforeseen, negative role for Cdt1 in elongation and suggest that its function in the control of replication should be redefined. We propose a novel surveillance mechanism in which Cdt1 blocks nascent chain elongation after detecting illegitimate activation of the licensing system.
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http://dx.doi.org/10.1091/mbc.e08-06-0613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633378PMC
February 2009

RecQ family helicases in genome stability: lessons from gene disruption studies in DT40 cells.

Cell Cycle 2008 Aug 18;7(16):2472-8. Epub 2008 Aug 18.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.

Cells of all living organisms have evolved complex mechanisms to maintain genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during DNA replication. RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. In human cells, there exist five RecQ DNA helicases, and mutations of three of these helicases, encoded by the BLM, WRN and RECQL4 genes, give rise to the cancer predisposition disorders, Bloom syndrome (BS), Werner syndrome (WS) and Rothmund-Thomson syndrome (RTS), respectively. Individuals suffering from WS and RTS also show premature aging phenotypes. Although the two remaining helicases, RECQL1 and RECQL5, have not yet been associated with heritable human diseases, a single nucleotide polymorphism of RECQL1 is associated with reduced survival of pancreatic cancer, and RecQl5 knockout mice show a predisposition to cancer. Here, we review the functions of eukaryotic RecQ helicases, focusing primarily on BLM in the maintenance of genome stability through various pathways of nucleic acid metabolism and with special reference to DNA replication.
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http://dx.doi.org/10.4161/cc.7.16.6462DOI Listing
August 2008

KU70/80, DNA-PKcs, and Artemis are essential for the rapid induction of apoptosis after massive DSB formation.

Cell Signal 2008 Nov 10;20(11):1978-85. Epub 2008 Jul 10.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.
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http://dx.doi.org/10.1016/j.cellsig.2008.07.006DOI Listing
November 2008

Rad52 sumoylation and its involvement in the efficient induction of homologous recombination.

DNA Repair (Amst) 2008 Jun 8;7(6):879-89. Epub 2008 Apr 8.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

The protein Rad52 is a key player in various types of homologous recombination and is essential to maintenance of genomic integrity. Although evidence indicates that Rad52 is modified by SUMO, the physiological relevance of this sumoylation remains unclear. Here, we identify the conditions under which Rad52 sumoylation is induced, and clarify the role of this modification in homologous recombination. Oligomerization of Rad52 was a prerequisite for sumoylation, and the modification occurred in the cell proceeding S phase being exposed to the DNA-damaging agent methyl methanesulfonate (MMS). Following exposure to MMS, sumoylated Rad52 accumulated in rad51 cells, but not in the recombination-related gene mutants, rad54, rad55, rad59, sgs1, or srs2. The accumulation of sumoylated Rad52 was suppressed in rad51 cells expressing Rad51-K191R, an ATPase-defective protein presumed to be recruited to ssDNA. Although the sumoylation defective mutant rad52-3KR (K10R/K11R/K220R) showed no defect in mating-type switching, which did not lead to Rad52 sumoylation in wild-type cells, the mutant did demonstrate a partial defect in MMS-induced interchromosomal homologous recombination.
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http://dx.doi.org/10.1016/j.dnarep.2008.02.005DOI Listing
June 2008

Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability.

Genes Genet Syst 2008 Feb;83(1):95-100

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan.

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.
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http://dx.doi.org/10.1266/ggs.83.95DOI Listing
February 2008

Insight into initiator-DNA interactions: a lesson from the archaeal ORC.

Bioessays 2008 Mar;30(3):208-11

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

Although initiation of DNA replication is considered to be highly coordinated through multiple protein-DNA and protein-protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences. Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins.
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http://dx.doi.org/10.1002/bies.20726DOI Listing
March 2008

Analyses of functional interaction between RECQL1, RECQL5, and BLM which physically interact with DNA topoisomerase IIIalpha.

Biochim Biophys Acta 2008 Feb 22;1782(2):75-81. Epub 2007 Nov 22.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai, Japan.

RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3alpha whose defect is lethal for the cell. Therefore in this study, we characterized recql5/recql1/blm triple mutants from DT40 cells to determine whether the triple mutants show a top3alpha disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm/recql1 and recql5/blm cells, and recql5/recql1/blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5/blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5/blm cells.
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http://dx.doi.org/10.1016/j.bbadis.2007.11.003DOI Listing
February 2008

Functional interactions between BLM and XRCC3 in the cell.

J Cell Biol 2007 Oct;179(1):53-63

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Science, Tohoku University, Sendai 980-8578, Japan.

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.
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http://dx.doi.org/10.1083/jcb.200702183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064734PMC
October 2007

Ctf18 is required for homologous recombination-mediated double-strand break repair.

Nucleic Acids Res 2007 18;35(15):4989-5000. Epub 2007 Jul 18.

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba 6-3, Aramaki, Aoba-ku, Sendai 980-8578, Japan.

The efficient repair of double-strand breaks (DSBs) is crucial in maintaining genomic integrity. Sister chromatid cohesion is important for not only faithful chromosome segregation but also for proper DSB repair. During DSB repair, the Smc1-Smc3 cohesin complex is loaded onto chromatin around the DSB to support recombination-mediated DSB repair. In this study, we investigated whether Ctf18, a factor implicated in the establishment of sister chromatid cohesion, is involved in DSB repair in budding yeast. Ctf18 was recruited to HO-endonuclease induced DSB sites in an Mre11-dependent manner and to damaged chromatin in G2/M phase-arrested cells. The ctf18 mutant cells showed high sensitivity to DSB-inducible genotoxic agents and defects in DSB repair, as well as defects in damage-induced recombination between sister chromatids and between homologous chromosomes. These results suggest that Ctf18 is involved in damage-induced homologous recombination.
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http://dx.doi.org/10.1093/nar/gkm523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976461PMC
October 2007

Function of recQ family helicase in genome stability.

Subcell Biochem 2006 ;40:49-73

Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan.

The recQ gene of Escherichia coli is the founding member of the RecQ family of helicases. Like E. coli, lower eukaryotic species also possess single RecQ proteins, such as Sgs1 and Rqh1 in budding and fission yeast, respectively. However, there are five RecQ helicases in human as well as in chicken cells. Three of the human RecQ helicases are encoded by BLM, WRN and RECQL4 genes, defects of which give rise to the cancer predisposition disorders known as Bloom syndrome (BS), Werner syndrome (WS) and Rothmund-Thomson syndrome (RTS), respectively. The other two, RECQL1 and RECQL5, have not been associated with human diseases. Characterization of RecQ family proteins in unicellular organisms has revealed that their defects confer genomic instability and impairment of homologous recombination. Although systematic genetic analysis of human BS, WS, and RTS cells must be useful to understand their functions, such approach is hampered by the difficulty of making cell lines with double gene disruptions. In this context, the chicken DT40 cell line is an ideal experimental tool for sophisticated approaches that illuminate the functions of vertebrate RecQ helicases. Here, we briefly review general features of RecQ helicases and describe their functions as revealed by analysis of DT40 cells.
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http://dx.doi.org/10.1007/978-1-4020-4896-8_5DOI Listing
July 2007
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