松浦 彰

In Saccharomyces cerevisiae, telomere replication occurs in late S phase and is accompanied by dynamic remodeling of its protein components. Here, we show that MRX (Mre11-Rad50-Xrs2), an evolutionarily conserved protein complex involved in DNA double-strand break (DSB) repair, is recruited to the telomeres in late S phase. MRX is required for the late S phase-specific recruitment of ATR-like kinase Mec1 to the telomeres. Mec1, in turn, contributes to the assembly of the telomerase regulators Cdc13 and Est1 at the telomere ends. Our results provide a model for the hierarchical assembly of telomere-replication proteins in late S phase; this involves triggering by the loading of MRX onto the chromosome termini. The recruitment of DNA repair-related proteins to the telomeres at particular times in the cell cycle suggests that the normal terminus of a chromosome is recognized as a DSB during the course of replication.

ATM and rad3-related protein kinase (ATR), a member of the phosphoinositide kinase-like protein kinase family, plays a critical role in cellular responses to DNA structural abnormalities in conjunction with its interacting protein, ATRIP. Here, we show that the amino-terminal portion of ATRIP is relocalized to DNA damage-induced nuclear foci in an RPA-dependent manner, despite its lack of ability to associate with ATR. In addition, ATR-free ATRIP protein can be recruited to the nuclear foci. Our results suggest that the N-terminal domain of the ATRIP protein contributes to the cell cycle checkpoint by regulating the intranuclear localization of ATR. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

PI-kinase-related protein kinase ATR forms a complex with ATRIP and plays pivotal roles in maintaining genome integrity. When DNA is damaged, the ATR-ATRIP complex is recruited to chromatin and is activated to transduce the checkpoint signal, but the precise kinase activation mechanism remains unknown. Here, we show that ATRIP is phosphorylated in an ATR-dependent manner after genotoxic stimuli. The serine 68 and 72 residues are important for the phosphorylation in vivo and are required exclusively for direct modification by ATR in vitro. Using phospho-specific antibody, we demonstrated that phosphorylated ATRIP accumulates at foci induced by DNA damage. Moreover, the loss of phosphorylation does not lead to detectable changes in the relocalization of ATRIP to nuclear foci nor in the activation of downstream effector proteins. Collectively, our results suggest that the ATR-mediated phosphorylation of ATRIP at Ser-68 and -72 is dispensable for the initial response to DNA damage. (C) 2004 Elsevier Inc. All rights reserved.

The phosphoinositide (PI)-3-kinase-related kinase (PIKK) family proteins Tel1p and Mec1p have been implicated in the telomere integrity of Saccharomyces cerevisiae. However, the mechanism of PIKK-mediated telomere length control remains unclear. Here, we show that Tel1p and Mec1p are recruited to the telomeres at specific times in the cell cycle in a mutually exclusive manner. In particular, Mec1p interacts with the telomeres during late S phase and is associated preferentially with shortened telomeres. We propose a model in which telomere integrity is maintained by the reciprocal association of PIKKs, and Mec1p acts as a sensor for structural abnormalities in the telomeres. Our study suggests a mechanistic similarity between telomere length regulation and DNA double-strand break repair, both of which are achieved by the direct association of PIKKs.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination and repair. In Saccharomyces cerevisiae, several mutants in the RFA1 gene encoding the large subunit of RPA have been isolated and one of the mutants with a missense allele, rfa1-D228Y, shows a synergistic reduction in telomere length when combined with a yku70 mutation. So far, only one mutant allele of the rad11(+) gene encoding the large subunit of RPA has been reported in Schizosaccharomyces pombe. To study the role of S.pombe RPA in DNA repair and possibly in telomere maintenance, we constructed a rad11-D223Y mutant, which corresponds to the S.cerevisiae rfa1-D228Y mutant. rad11-D223Y cells were methylmethane sulfonate, hydroxyurea, UV and gamma-ray sensitive, suggesting that rad11-D223Y cells have a defect in DNA repair activity. Unlike the S.cerevisiae rfa1-D228Y mutation, the rad11-D223Y mutation itself caused telomere shortening. Moreover, Rad11-Myc bound to telomere in a ChIP assay. These results strongly suggest that RPA is directly involved in telomere maintenance.

The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism. In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT). Here we report a novel mammalian protein homologous to CACT. The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity. The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed. In addition, CACL is induced by partial hepatectomy or fasting. Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism. In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT). Here we report a novel mammalian protein homologous to CACT. The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity. The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed. In addition, CACL is induced by partial hepatectomy or fasting. Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function.

The Schizosaccharomyces pombe Ku70-Ku80 heterodimer is required for telomere length regulation. Lack of pku70(+) results in telomere shortening and striking rearrangements of telomere-associated sequences. We found that the rearrangements of telomere-associated sequences in pku80(+) mutants are Rhp51 dependent, but not Rad50 dependent. Rhp51 bound to telomere ends when the Ku heterodimer was not present at telomere ends. We also found that the single-stranded G-rich tails increased in S phase in wild-type strains, while deletion of pku70(+) increased the single-stranded overhang in both G(2) and S phase. Based on these observations, we propose that Rhp51 binds to the G-rich overhang and promotes homologous pairing between two different telomere ends in the absence of Ku heterodimer. Moreover, pku80 rhp51 double mutants showed a significantly reduced telomere hybridization signal. Our results suggest that, although Ku heterodimer sequesters Rhp51 from telomere ends to inhibit homologous recombination activity, Rhp51 plays important roles for the maintenance of telomere ends in the absence of the Ku heterodimer.

The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methane sulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70(+). However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70(+). The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50(+), but the overhang reappeared following concomitant deletion of pku70(+). Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.

Background. Liver regeneration after partial hepatectomy (PH) is accomplished by a synchronous replication of hepatocytes. Both positive and negative regulators of cyclin-dependent protein kinase (Cdk) have been implicated in hepatocyte proliferation, but their specific roles in vivo remain to be clarified. To investigate the specific role of p27(Kip1), a member of the Cip/Kip family of Cdk inhibitors, in cell-cycle regulation during liver regeneration, p27-knockout mice were studied after PH. Materials and methods. Under ether anesthesia, mice were subjected to 70% PH. Animals were sacrificed at intervals after the surgery, and the remnant liver was harvested and analyzed. Results. In p27-deficient mice, the timing of DNA synthesis was significantly accelerated with a perturbation in the ordered distribution of proliferating cells in the hepatic lobule. p27 deficiency, however, did not affect the whole population of cycling cells, the number of apoptotic cells, or liver injury and mortality after PH. Conclusion. These data provide in vivo evidence that p27 functions as a brake in the "start" of the hepatocyte cell cycle, thereby coordinating temporally and spatially the onset of DNA synthesis of hepatocytes within the hepatic lobules. (C) 2003 Elsevier Inc. All rights reserved.

Background. Liver regeneration after partial hepatectomy (PH) is accomplished by a synchronous replication of hepatocytes. Both positive and negative regulators of cyclin-dependent protein kinase (Cdk) have been implicated in hepatocyte proliferation, but their specific roles in vivo remain to be clarified. To investigate the specific role of p27(Kip1), a member of the Cip/Kip family of Cdk inhibitors, in cell-cycle regulation during liver regeneration, p27-knockout mice were studied after PH. Materials and methods. Under ether anesthesia, mice were subjected to 70% PH. Animals were sacrificed at intervals after the surgery, and the remnant liver was harvested and analyzed. Results. In p27-deficient mice, the timing of DNA synthesis was significantly accelerated with a perturbation in the ordered distribution of proliferating cells in the hepatic lobule. p27 deficiency, however, did not affect the whole population of cycling cells, the number of apoptotic cells, or liver injury and mortality after PH. Conclusion. These data provide in vivo evidence that p27 functions as a brake in the "start" of the hepatocyte cell cycle, thereby coordinating temporally and spatially the onset of DNA synthesis of hepatocytes within the hepatic lobules. (C) 2003 Elsevier Inc. All rights reserved.

Relocation into the nucleus of the yeast cytoplasmic linear plasmids was studied using a monitor plasmid pCLU1. In Saccharomyces cerevisiae, the nuclearly-relocated pCLU1 replicated in a linear form (termed pTLU-type plasmid) which carried the host telomeric repeats TG1-3 of 300-350 bp at both ends. The telomere sequences mainly consisted of a major motif TGTGTGGGTGTGG which was complementary to part of the RNA template of yeast telomerase and were directly added to the very end of the pCLU1-terminal element ITR (inverted terminal repeat), suggesting that the ITR end played a role as a substrate of telomerase. The telomere sequences varied among isolated pTLU-type plasmids, but the TG1-3 organization was symmetrically identical on both ends of any one plasmid. During cell growth under non-selective condition, the telomeric repeat sequences were progressively rearranged on one side, but not on the opposite side of pTLU plasmid ends. This indicates that the mode of telomeric DNA replication or repair differed between both ends. Clonal analysis showed that the intense rearrangement of telomeric DNA was closely associated with extreme instability of pTLU plasmids.

Autophagy is a cellular process for bulk degradation of cytoplasmic components. The attachment of Apg12p, a modifier with no significant similarity to ubiquitin, to Apg5p is crucial for autophagy in yeast, This reaction proceeds in a ubiquitination-like manner, and requires Apg7p and Apg10p, Apg7p exhibits a considerable similarity to ubiquitin-activating enzyme (E1) and is found to activate Apg12p with ATP hydrolysis. Apg10p, on the other hand, shows no significant similarity to other proteins whose functions are known. Here, we show that after activation by Apg7p, Apg12p is transferred to the Cys-133 residue of Apg10p to form an Apg12p-Apg10p thioester, Cells expressing Apg10p(C133S) do not generate the Apg12p-Apg5p conjugate, which leads to defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These findings indicate that Apg10p is a new type of protein-conjugating enzyme that functions in the Apg12p-Apg5D conjugation pathway.

The Schizosaccharomyces pombe checkpoint gene named rad3(+) encodes an ATM-homologous protein kinase that shares a highly conserved motif with proteins involved in DNA metabolism. Previous studies have shown that Rad3 fulfills its function via the regulation of the Chk1 and Cds1 protein kinases. Here we describe a novel role for Rad3 in the control of telomere integrity. Mutations in the rad3(+) gene alleviated telomeric silencing and produced shortened lengths in the telomere repeat tracts. Genetic analysis revealed that the other checkpoint rad mutations rad1, rad17, and rad26 belong to the same phenotypic class with rad3 with regard to control of the telomere length. Of these mutations, rad3 and rad26 have a drastic effect on telomere shortening, tel1(+), another ATM homologue in S. pombe, carries out its telomere maintenance function in parallel with the checkpoint rad genes. Furthermore, either a single or double disruption of cds1(+) and chk1(+) caused no obvious changes in the telomeric DNA structure. Our results demonstrate a novel role of the S. pombe ATM homologues that is independent of chk1(+) and cds1(+).

The Saccharomyces cerevisiae STT3 (ScSTT3) gene encodes a protein which is involved in protein glycosylation via the regulation of oligosaccharyltransferase activity. We have cloned and isolated the Schizosaccharomyces pombe STT3 homologous gene (Spstt3(+)). The Spstt3(+) gene encodes a protein consisting of 749 amino acid residues which has significant homology with ScStt3p and the mouse Stt3p-homologue Itm1p. Disruption of the Spstt3(+) gene shows that this gene is essential for growth. Like Itm1, Spstt3(+) partially suppressed the temperature sensitivity of the stt3-1 mutation of S. cerevisiae, indicating that Spstt3(+) is a functional and structural homologue of the ScSTT3 gene. The nucleotide sequence data reported in this paper appears in the DDBJ/EMBL/GenBank nucleotide sequence database with Accession No. AB015232. Copyright (C) 1999 John Wiley & Sons, Ltd.

The yeast Y' element is a highly polymorphic repetitive sequence present in the subtelomeric regions of many yeast telomeres. The Y' element is classed as either Y'-L or Y'-S, depending on its length. It has been reported that survivors arising from telomerase-deficient yeast mutants compensate for telomere loss by the amplification of Y' elements. The total Saccharomyces cerevisiae genome DNA data base was searched for Y' elements, and 11 Y'-Ls and eight Y'-Ss were identified. As reported previously, many of the sequences were found to contain long open reading frames which potentially encode helicase. We examined the expression of the Y' elements in telomerase-deficient Delta tlc1 survivors, in which the TLC1 gene encoding the yeast telomerase template RNA had been disrupted, and found that the Y' element is highly expressed in the survivors, but not in the wild-type cells. Moreover, we demonstrated that the survivors produce a Y'-encoded protein designated as Y'-Help1 (Y'-helicase protein 1), and that this protein possesses helicase activity. Therefore, we suggest that the Y' element has a novel and potentially important role in trans, in addition to the well characterized role in cis, in telomerase-independent telomere maintenance in yeast.

Telomeres, found at chromosomal ends, are essential for stable maintenance of linear chromosomes in eukaryotes. The ATM family of genes, including budding yeast TEL1 (refs 1,2), fission yeast rad3(+) (ref. 3) and human ATM (ref. 4), have been reported to be involved in telomere length regulation(5-7), although the significance of the telomere phenotypes observed with the mutated genes remains elusive. We have cloned tel1(+), another fission yeast ATM homologue, and found that a tel1rad3 double mutant lost all telomeric DNA sequences. Thus, the ATM homologues are essential in telomere maintenance. The mutant grew poorly and formed irregular-shaped colonies, probably due to chromosome instability, however, during prolonged culture of the double mutant, cells forming normal round-shaped colonies arose at a relatively high frequency. All three chromosomes in these derivative cells were circular and lacked telomeric sequences. To our knowledge, this is the first report of eukaryotic cells whose chromosomes are all circular. Upon meiosis, these derivative cells produced few viable spores. Therefore, the exclusively circular genome lacking telomeric sequences is proficient for mitotic growth, but does not permit meiosis.

Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles. We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation. Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase. Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process. We found overall homology of Apg1p with C. elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow. (C) 1997 Elsevier Science B.V.

We have isolated 14 apg mutants defective in autophagy in yeast Saccharomyces cerevisiae (Tsukada and Ohsumi, 1993). Among them, APG1 encodes a novel Ser/Thr protein kinase whose kinase activity is essential for autophagy. In the course of searching for genes that genetically interact with APG1, we found that overexpression of APG1 under control of the GAL1 promoter suppressed the autophagy-defective phenotype of apg13-1 mutant. Cloning and sequencing analysis showed that the APG13 gene encodes a novel hydrophilic protein of 738 amino acid residues. APG13 gene is constitutively expressed bot not starvation-inducible. Though dispensable for cell proliferation, APG13 is important for maintenance of cell viability under starvation conditions. apg13 disruptants were defective in autophagy like apg13-1 mutants. Morphological and biochemical investigation showed that a defect in autophagy of Delta apg13 was also suppressed by APG1 overexpression. These results imply genetic interaction between APG1 and APG13. (C) 1997 Elsevier Science B.V.

We have cloned and characterized the rat telomerase protein component 1 gene (TLP1), which is related to the gene for Tetrahymena p80. The cDNA encodes a 2629 amino acid sequence and produces the TLP1 proteins p240 and p230. The anti-TLP1 antibody specifically immunoprecipitated the telomerase activity. Moreover, p240 and p230 were copurified with telomerase activity in a series of extensive purification experiments. These results strongly suggest that the TLP1 proteins are components of, or are closely associated with, the rat telomerase. A pulse-chase experiment showed that p240 is modified to p230 in vivo. p230 was the dominant form in telomerase-positive cells, suggesting that modification of the TLP1 protein may regulate telomerase activity in vivo.

Acidification inside vacuoles has been shown to play a key role in a number of physiologically important cellular events. We studied the role of vacuolar membrane H+-ATPase in the autophagic process of Saccharomyces cerevisiae. Mutants lacking VMA genes which encode their subunits of the vacuolar H+-ATPase accumulated autophagic bodies in vacuoles on starvation. uma mutants also had a defect in protein degradation induced by starvation, In vma mutants, the activities of vacuolar proteases were remarkably lower than those of the wild-type. Overexpression of vacuolar proteases did not overcome the defect is the disintegration of autophagic bodies in vma mutant, even the overexpressed proteinase A and proteinase B being substantially localized to the vacuolar compartment and undergoing proper proteolytic maturation. Our results showed that the acidification of vacuoles is not required for the formation and delivery of autophagosomes to vacuoles, but is essential for the disintegration of autophagic bodies.

The APGS gene of Saccharomyces cerevisiae was cloned from a yeast genomic library by complementation of autophagy defective phenotype of apg5-1 mutant. structural analysis of the obtained genomic fragment showed that the APG5 gene encodes a novel hydrophilic protein of 294 amino-acid residues without apparent structural similarities to other proteins in the database. To examine its function, a null allele for APG5 (Delta apg5) was constructed and introduced into yeast. dapg5 cells germinated and grew normally in nutrient-rich condition, however, their viability reduced significantly upon the nutrient starvation. They were also shown to be defective in autophagy: they could not sequester autophagic bodies in the vacuole under nitrogen-starvation conditions. These phenotypes are identical to those found in the apg5-1 mutant. The lack of apparent phenotype in rich medium suggests that APGS function is required only under nutrient starvation condition, however, Northern blot analysis showed that its expression levels remained unchanged after nutrient depletion.

The yeast S. cerevisiae imports cytosolic components into the vacuole non-selectively by autophagy and degrades them by vacuolar hydrolases under nutrient starvation conditions. We developed a novel system for monitoring autophagy by constructing cells in which modified vacuolar alkaline phosphatase is expressed as an inactive precursor form in the cytosol. Under starvation conditions, the processing of the precursor to the mature form and phosphatase activity appeared gradually, and the mature form was located in the vacuole. Disruption of APG1, an essential gene for autophagy, resulted in no processing or phosphatase activity. These results indicate that the precursor form in the cytosol is transferred to the vacuole by autophagy and converted to the active form by vacuolar proteinases. Thus, autophagy could be determined easily and accurately by measuring the phosphatase activity. (C) 1995 Academic Press, Inc.

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1(ts) mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.

In order to isolate genes that function downstream of the Ras-cAMP pathway in Saccharomyces cerevisiae, a YEp13-based genomic library was screened for clones that inhibit growth of cells with diminished A-kinase activity. One such gene, MKS1, was found to encode a hydrophilic 52 kDa protein that shares weak homology with the yeast SPT2/SIN1 gene product. Three lines of evidence suggest that the MKS1 gene product is a negative regulator downstream of the Ras-cAMP pathway: (i) overexpression of MKS1 inhibits growth of cyr1 disruptant cells on YPD medium containing a low concentration of cAMP; (ii) overexpression of MKS1 does not affect TPK1 expression; and (iii) the temperature-sensitive cyr1-230 mutation is partially suppressed by mks1 disruption. The mks1 mutant shows similar phenotypes to gal11/spt13, i.e., it cannot grow on YPGal containing ethidium bromide at 25-degrees-C, or on YPGly or SGal at 37-degrees-C. The mks1 gal11 double mutant shows more marked phenotypic changes than the single mutants. These results suggest that MKS1 is involved in transcriptional regulation of several genes by cAMP.

cyr1-2 is a temperature-sensitive mutation of the yeast adenylate cyclase structural gene, CYR1. The cyr1-2 mutation has been suggested to be a UGA mutation since a UGA suppressor SUP201 has been isolated as a suppressor of the cyr1-2 mutation. Construction of chimeric genes restricted the region containing the cyr1-2 mutation, and the cyr1-2 UGA mutation was identified at codon 1282, which lies upstream of the region coding for the catalytic domain of adenylate cyclase. Alterations in the region upstream of the cyr1-2 mutation site result in null mutations. The complete open reading frame of the cyr1-2 gene expressed under the control of the GAL1 promoter complemented cyr1-d1 in a galactose-dependent manner. These results suggest that at the permissive temperature weak readthrough occurs at the cyr1-2 mutation site to produce low levels of active adenylate cyclase. An endogenous suppressor in yeast cells is assumed to be responsible for this readthrough.