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Biomedical subjects

A Sugino

Publications and source records attributed to A Sugino.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis.

The initiation of DNA synthesis is an important cell cycle event that defines the beginning of S phase. This critical event involves the participation of proteins whose functions are regulated by cyclin dependent protein kinases (Cdks). The Mcm2-7 proteins are a family of six conserved proteins that are essential for the initiation of DNA synthesis in all eukaryotes. In Saccharomyces cerevisiae, members of the Mcm2-7 family undergo cell cycle-specific phosphorylation. Phosphorylation of Mcm proteins at the beginning of S phase coincides with the removal of these proteins from chromatin and the onset of DNA synthesis. In this study, we identified DBF4, which encodes the regulatory subunit of a Cdk-like protein kinase Cdc7-Dbf4, in a screen for second site suppressors of mcm2-1. The dbf4 suppressor mutation restores competence to initiate DNA synthesis to the mcm2-1 mutant. Cdc7-Dbf4 interacts physically with Mcm2 and phosphorylates Mcm2 and three other members of the Mcm2-7 family in vitro. Blocking the kinase activity of Cdc7-Dbf4 at the G1-to-S phase transition also blocks the phosphorylation of Mcm2 at this defined point of the cell cycle. Taken together, our data suggest that phosphorylation of Mcm2 and probably other members of the Mcm2-7 proteins by Cdc7-Dbf4 at the G1-to-S phase transition is a critical step in the initiation of DNA synthesis at replication origins.

Cell Cycle Proteins↗
PubMed1996

Rfc5, a small subunit of replication factor C complex, couples DNA replication and mitosis in budding yeast.

The inhibition of DNA synthesis prevents mitotic entry through the action of the S phase checkpoint. In the yeast Saccharomyces cerevisiae, an essential protein kinase, Spk1/Mec2/Rad53/Sad1, controls the coupling of S phase to mitosis. In an attempt to identify genes that genetically interact with Spk1, we have isolated a temperature-sensitive mutation, rfc5-1, that can be suppressed by overexpression of SPK1. The RFC5 gene encodes a small subunit of replication factor C complex. At the restrictive temperature, rfc5-1 mutant cells entered mitosis with unevenly separated or fragmented chromosomes, resulting in loss of viability. Thus, the rfc5 mutation defective for DNA replication is also impaired in the S phase checkpoint. Overexpression of POL30, which encodes the proliferating cell nuclear antigen, suppressed the replication defect of the rfc5 mutant but not its checkpoint defect. Taken together, these results suggested that replication factor C has a direct role in sensing the state of DNA replication and transmitting the signal to the checkpoint machinery.

Amino Acid Sequence↗
PubMed1995

Dpb11, which interacts with DNA polymerase II(epsilon) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint.

DPB11, a gene that suppresses mutations in two essential subunits of Saccharomyces cerevisiae DNA polymerase II(epsilon) encoded by POL2 and DPB2, was isolated on a multicopy plasmid. The nucleotide sequence of the DPB11 gene revealed an open reading frame predicting an 87-kDa protein. This protein is homologous to the Schizosaccharomyces pombe rad4+/cut5+ gene product that has a cell cycle checkpoint function. Disruption of DPB11 is lethal, indicating that DPB11 is essential for cell proliferation. In thermosensitive dpb11-1 mutant cells, S-phase progression is defective at the nonpermissive temperature, followed by cell division with unequal chromosomal segregation accompanied by loss of viability.dpb11-1 is synthetic lethal with any one of the dpb2-1, pol2-11, and pol2-18 mutations at all temperatures. Moreover, dpb11 cells are sensitive to hydroxyurea, methyl methanesulfonate, and UV irradiation. These results strongly suggest that Dpb11 is a part of the DNA polymerase II complex during chromosomal DNA replication and also acts in a checkpoint pathway during the S phase of the cell cycle to sense stalled DNA replication.

Amino Acid Sequence↗
PubMed1995

Yeast DNA polymerases and their role at the replication fork.

The DNA polymerases of the yeast Saccharomyces cerevisiae serve as a model system for the study of the replication fork during DNA replication. To date, six S. cerevisiae DNA polymerases have been at least partially characterized (compared with four in mammals so far), with further candidates being identified as open reading frames in the yeast genome sequencing project. Here, we review the current state of knowledge of the yeast polymerases, and discuss, where possible, their biological role during DNA replication.

DNA Polymerase II↗
PubMed1995

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Base Sequence↗
PubMed1994

The yeast Saccharomyces cerevisiae DNA polymerase IV: possible involvement in double strand break DNA repair.

We identified and purified a new DNA polymerase (DNA polymerase IV), which is similar to mammalian DNA polymerase beta, from Saccharomyces cerevisiae and suggested that it is encoded by YCR14C (POLX) on chromosome III. Here, we provided a direct evidence that the purified DNA polymerase IV is indeed encoded by POLX. Strains harboring a pol4 deletion mutation exhibit neither mitotic growth defect nor a meiosis defect, suggesting that DNA polymerase IV participates in nonessential functions in DNA metabolism. The deletion strains did not exhibit UV-sensitivity. However, they did show weak sensitivity to MMS-treatment and exhibited a hyper-recombination phenotype when intragenic recombination was measured during meiosis. Furthermore, MAT alpha pol4 delta segregants had a higher frequency of illegitimate mating with a MAT alpha tester strain than that of wild-type cells. These results suggest that DNA polymerase IV participates in a double-strand break repair pathway. A 3.2kb of the POL4 transcript was weakly expressed in mitotically growing cells. During meiosis, a 2.2 kb POL4 transcript was greatly induced, while the 3.2 kb transcript stayed at constant levels. This induction was delayed in a swi4 delta strain during meiosis, while no effect was observed in a swi6 delta strain.

Amino Acid Sequence↗
PubMed1994

The RFC2 gene encoding a subunit of replication factor C of Saccharomyces cerevisiae.

Replication Factor C (RF-C) of Saccharomyces cerevisiae is a complex that consists of several different polypeptides ranging from 120- to 37 kDa (Yoder and Burgers, 1991; Fien and Stillman, 1992), similar to human RF-C. We have isolated a gene, RFC2, that appears to be a component of the yeast RF-C. The RFC2 gene is located on chromosome X of S. cerevisiae and is essential for cell growth. Disruption of the RFC2 gene led to a dumbbell-shaped terminal morphology, common to mutants having a defect in chromosomal DNA replication. The steady-state levels of RFC2 mRNA fluctuated less during the cell cycle than other genes involved in DNA replication. Nucleotide sequence of the gene revealed an open reading frame corresponding to a polypeptide with a calculated Mr of 39,716 and a high degree of amino acid sequence homology to the 37-kDa subunit of human RF-C. Polyclonal antibodies against bacterially expressed Rfc2 protein specifically reduced RF-C activity in the RF-C-dependent reaction catalyzed by yeast DNA polymerase III. Furthermore, the Rfc2 protein was copurified with RF-C activity throughout RF-C purification. These results strongly suggest that the RFC2 gene product is a component of yeast RF-C. The bacterially expressed Rfc2 protein preferentially bound to primed single-strand DNA and weakly to ATP.

Amino Acid Sequence↗
PubMed1994

The 3'-->5' exonucleases of both DNA polymerases delta and epsilon participate in correcting errors of DNA replication in Saccharomyces cerevisiae.

DNA polymerases II (epsilon) and III (delta) are the only nuclear DNA polymerases known to possess an intrinsic 3'-->5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase delta and epsilon 3'-->5' exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10(2) and 10(1), respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 x 10(3)-fold. Analysis of mutation rates of double mutants indicated that the 3'-->5' exonucleases of DNA polymerases delta and epsilon can act competitively and that, like the 3'-->5' exonuclease of DNA polymerase delta, the 3'-->5' exonuclease of DNA polymerase epsilon acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3'-->5' exonucleases of DNA polymerases delta and epsilon act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.

Amino Acid Sequence↗
PubMed1994

Molecular analysis of the dhp1+ gene of Schizosaccharomyces pombe: an essential gene that has homology to the DST2 and RAT1 genes of Saccharomyces cerevisiae.

The DST2 gene of Saccharomyces cerevisiae encodes a DNA strand exchange protein, STP beta, which is required for homologous recombination in both mitotic or meiotic cells. We have cloned a DST2-related gene from the fission yeast Schizosaccharomyces pombe and designated it dhp1+. The nucleotide sequence of dhp1+ revealed an open reading frame encoding a protein composed of 991 amino acids. The predicted amino acid sequence was significantly homologous to the S. cerevisiae STP beta, but lacked the carboxy-terminal sequence present in STP beta. Furthermore, dhp1+ shows greater homology to RAT1/HKE1, a gene which is involved in RNA trafficking and processing. Genetic experiments showed that dhp1+ on an S. cerevisiae expression vector could rescue both the defects of the S. cerevisiae DST2 disruptant, slow growth rate and a sporulation defect, and the lethality of the S. cerevisiae rat1ts mutation. This implies the functional similarity of dhp1+ to both DST2 and RAT1. However unlike DST2, dhp1+ is an essential gene for cell growth in S. pombe, suggesting that dhp1+ is not the true homologue of DST2 but rather of RAT1 in S. pombe. The possible roles of dhp1+ in recombination and cell growth in S. pombe are discussed.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta.

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.

Animals↗
PubMed1993

Purification and characterization of DNA helicase III from the yeast Saccharomyces cerevisiae.

Two forms of DNA helicase activity, Rad3 and ATPase III, were previously purified from the yeast Saccharomyces cerevisiae and characterized. Here, we have identified and purified an additional DNA helicase activity from S. cerevisiae to near homogeneity. This helicase differs from those described previously in its chromatographic behavior, molecular weight, enzymatic properties, and genetic properties. Thus, we named it DNA helicase III. Its apparent molecular mass is about 120 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. DNA helicase III requires a divalent cation Mg2+ or Mn2+, either ATP or dATP, and a single-stranded portion on the duplex substrate. Helicase III moves in the 5'-->3' direction on single-stranded portions of the substrate and unwinds the strand of DNA in the 3'-->5' direction. It also has an intrinsic DNA-dependent ATPase (dATPase) activity that hydrolyzes either ATP or dATP to ADP or dADP and orthophosphate in the presence of DNA. DNA helicase III activity was not affected by either rad3 or radH mutations, suggesting that it is encoded by a gene different from RAD3 and RADH.

Adenosine Triphosphate↗
PubMed1993

Pathway correcting DNA replication errors in Saccharomyces cerevisiae.

Mutation of predicted 3'-->5' exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease-deficient mutant pol3-01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3-01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3-01/pol3-01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 x 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3-01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co-regulated with these genes. We propose that the POL3 3'-->5' exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ-->mutHLS pathway of DNA replication error correction in Escherichia coli.

Amino Acid Sequence↗
PubMed1993

A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5.

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. Nucleotide sequence analysis of the suppressor gene, provisionally named MSD2, revealed an open reading frame encoding a protein with a calculated M(r) of 81,024, with amino acid sequence similarity to the catalytic domains of protein kinases. Both genetic linkage and complementation analyses indicated that MSD2 is identical to the cell division cycle gene CDC5. An activity that phosphorylated exogenously added casein was immunoprecipitated by antiserum against a TrpE-Cdc5 fusion protein from lysates of wild-type cells containing CDC5 on a multicopy plasmid but not of cells bearing a small deletion in the predicted protein kinase domain of CDC5 on the plasmid. Deletion of CDC5 was lethal and resulted in a dumbbell-shaped terminal morphology, with the nuclei almost divided but still connected. Consistent with the function at the G2/M boundary, the CDC5 transcript accumulated periodically during the cell cycle, peaking at the G2/M boundary. CDC5 on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc20, and dbf2 mutations which affect mitosis during the cell cycle.

Alleles↗
PubMed1992

Reduction and inactivation of the sarcoplasmic Ca(2+)-ATPase by 2-mercaptoethanol--contrast to the (Na+,K+) ATPase.

The sarcoplasmic Ca(2+)-ATPase was reduced with 300 mM 2-mercaptoethanol at elevated temperatures (40-45 degrees C) with a concomitant loss of ATPase activity. The reduction and inactivation of the Ca(2+)-ATPase proceeded rapidly in the absence of Ca2+. The Ca(2+)-ATPase was also inactivated with 2-mercaptoethanol in the presence of diluted SDS (0.4 mg/ml) even at 20 degrees C. In contrast to the (Na+, K+) ATPase, the inactivated Ca(2+)-ATPase in the presence of diluted SDS was sedimented by the centrifugation at 100,000 x g for 30 min.

Animals↗
PubMed1992

DNA polymerase II, the probable homolog of mammalian DNA polymerase epsilon, replicates chromosomal DNA in the yeast Saccharomyces cerevisiae.

Two temperature-sensitive DNA polymerase II mutants (pol2-9 and pol2-18) of the yeast Saccharomyces cerevisiae were isolated by the plasmid shuffling method. DNA polymerase II activity partially purified from both mutants was thermolabile, while DNA polymerase I and III activities remained thermotolerant. At the restrictive temperature, the pol2 mutants were defective in chromosomal DNA replication and exhibited the dumbbell terminal morphology typical of DNA replication mutants. The POL2 transcript accumulated periodically during the cell cycle, peaking at the G1/S boundary in the same manner as the transcripts of more than 10 other DNA replication genes. These results indicate that DNA polymerase II participates in nuclear DNA replication. The similarities in structure and activities between the DNA polymerases of yeast and mammals make it likely that mammalian DNA polymerase epsilon too is required for chromosomal DNA replication.

Amino Acid Sequence↗