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B K Tye

Publications and source records attributed to B K Tye.

At least 19 recordsLinked to original sources
PubMed2001

Initiating DNA synthesis: from recruiting to activating the MCM complex.

The exact duplication of a genome once per cell division is required of every proliferating cell. To achieve this goal, eukaryotes adopt a strategy that limits every replication origin to a single initiation event within a narrow window of the cell cycle by temporally separating the assembly of the pre-replication complex (pre-RC) from the initiation of DNA synthesis. A key component of the pre-RC is the hexameric MCM complex, which is also the presumed helicase of the growing forks. An elaborate mechanism recruits the MCM complex to replication origins, and a regulatory chain reaction converts the poised, but inactive, MCM complex into an enzymatically active helicase. A growing list of proteins, including Mcm10 and Cdt1, are involved in the recruitment process. Two protein kinases, the Cdc7-Dbf4 kinase (DDK) and the cyclin-dependent kinase (CDK), trigger a chain reaction that results in the phosphorylation of the MCM complex and finally in the initiation of DNA synthesis. A composite picture from recent studies suggests that DDK is recruited to the pre-RC during G1 phase but must wait until S phase to phosphorylate the MCM complex. CDK is required for the recruitment of Cdc45 and other downstream components of the elongation machinery.

Animals↗
PubMed2000

Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins.

MCM2-7, a complex of six subunits, is an essential component of the prereplication chromatin that is assembled at Saccharomyces cerevisiae replication origins during G(1) phase. It is also believed to be the processive helicase at growing forks. To elucidate the action of MCM2-7 during the transition from initiation to elongation replication, we have focused our studies on Mcm10, a replication initiation protein that physically interacts with members of the MCM2-7 complex. We show that Mcm10 is a chromatin-associated protein that mediates the association of the MCM2-7 complex with replication origins. Furthermore, diminished interaction between Mcm10 and Mcm7, a subunit of the MCM2-7 complex, by a mutation in either Mcm10 or Mcm7 inhibits replication initiation. Surprisingly, a double mutant containing both the mcm10-1 and mcm7-1 (cdc47-1) alleles restores interaction between Mcm10 and Mcm7 and corrects all of the defects exhibited by each of the single mutants, including the stalling of replication forks at replication origins typically seen in mcm10-1 cells. This mutual compensation of defects between two independently isolated mutations is allele specific. These results suggest that Mcm10, like Mcm7, is a critical component of the prereplication chromatin and that interaction between Mcm10 and Mcm7 is required for proper replication initiation and prompt release of origin-bound factors.

Alleles↗
PubMed1999

Minichromosome maintenance as a genetic assay for defects in DNA replication.

Minichromosome maintenance (mcm) is an effective genetic assay for mutants defective in DNA replication. Two classes of mcm mutants have been identified using this screen: those that differentially affect the activities of certain autonomously replicating sequences (ARSs) and those that uniformly affect the activities of all ARSs. The ARS-specific MCM genes are essential for the initiation of DNA replication. Among these are members of the MCM2-7 family that encode subunits of the preinitiation complex and MCM10, whose gene product interacts with members of the Mcm2-7 proteins. Among the ARS-nonspecific MCM gene products are chromosome transmission factors. Refinement of this genetic assay as a screening tool and further analysis of existing mcm mutants may reveal new replication initiation proteins.

Chromosomal Proteins, Non-Histone↗
PubMed1999

MCM proteins in DNA replication.

The MCM proteins are essential replication initiation factors originally identified as proteins required for minichromosome maintenance in Saccharomyces cerevisiae. The best known among them are a family of six structurally related proteins, MCM2-7, which are evolutionally conserved in all eukaryotes. The MCM2-7 proteins form a hexameric complex. This complex is a key component of the prereplication complex that assembles at replication origins during early G1 phase. New evidence suggests that the MCM2-7 proteins may be involved not only in the initiation but also in the elongation of DNA replication. Orchestration of the functional interactions between the MCM2-7 proteins and other components of the prereplication complex by cell cycle-dependent protein kinases results in initiation of DNA synthesis once every cell cycle.

Cell Cycle Proteins↗
PubMed1998

Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching.

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATalpha cells inhibit recombination along the entire left arm of chromosome III, including HML, whereas MATa cells activate this same region. MATa-dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATalpha cells by binding of the Matalpha2-Mcm1 corepressor to a site within the RE. Mutation of the two Matalpha2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATalpha cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATalpha cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matalpha2. Further, a mutation that alters the ability of Mcm1 to act with Matalpha2 in repressing a-specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matalpha2-Mcm1-mediated repression of RE activity.

Base Sequence↗
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↗
PubMed1997

Nuclear accumulation of Saccharomyces cerevisiae Mcm3 is dependent on its nuclear localization sequence.

BACKGROUND: The proteins of the Mcm2-7 family are required for the initiation of DNA replication. In Saccharomyces cerevisiae the nuclear envelope does not break down during the mitotic phase of the cell cycle. Large nuclear proteins, such as the Mcm proteins, which accumulate in the nucleus during specific portions of the cell cycle, must have regulated mechanisms to direct their entry into the nucleus. RESULTS: We have identified a nuclear localization sequence (NLS) in Mcm3, and demonstrated that it is necessary for the translocation of Mcm3 into the nucleus and sufficient for directing Escherichia coli beta-galactosidase to the nucleus. Immediately adjacent to the nuclear localization sequence are four potential sites for phosphorylation by Cdc28. Mutagenesis of all four sites has no immediate phenotypic effect on cell growth or viability, nor does it affect nuclear accumulation of Mcm3, although two-dimensional protein gel analysis has shown that at least some of these sites are normally phosphorylated in vivo. Substitution of the Mcm3 NLS by the SV40 large T-antigen NLS also directs the nuclear accumulation of the Mcm3-T-antigen protein, although cell growth is compromised. Replication activity in cells bearing either the Mcm3-Cdc28 phosphorylation site mutations or the Mcm3 T-antigen NLS substitution, as measured by plasmid stability assays, is comparable to activity in wild-type cells. CONCLUSIONS: The Mcm3 protein is imported into the nucleus by a specific NLS. The cell cycle specific nuclear accumulation of Mcm3 appears to be a result of nuclear retention or nuclear targeting, rather than nuclear import regulated through the NLS.

Amino Acid Sequence↗
PubMed1997

Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

Antibodies, Monoclonal↗
PubMed1997

A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae.

We describe a new minichromosome maintenance factor, Mcm10, and show that this essential protein is involved in the initiation of DNA replication in Saccharomyces cerevisiae. The mcm10 mutant has an autonomously replicating sequence-specific minichromosome maintenance defect and arrests at the nonpermissive temperature with dumbbell morphology and 2C DNA content. Mcm10 is a nuclear protein that physically interacts with several members of the MCM2-7 family of DNA replication initiation factors. Cloning and sequencing of the MCM10 gene show that it is identical to DNA43, a gene identified independently for its putative role in replicating DNA. Two-dimensional DNA gel analysis reveals that the mcm10-1 lesion causes a dramatic reduction in DNA replication initiation at chromosomal origins, including ORI1 and ORI121. Interestingly, the mcm10-1 lesion also causes replication forks to pause during elongation through these same loci. This novel phenotype suggests a unique role for the Mcm10 protein in the initiation of DNA synthesis at replication origins.

Alleles↗
PubMed1996

Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae.

Mcm2, Mcm3, and Mcm5/Cdc46 are conserved proteins essential for the initiation of DNA synthesis at replication origins in Saccharomyces cerevisiae. The accumulation of these proteins in the nucleus before the onset of DNA synthesis suggests that they play a role in restricting DNA synthesis to once per cell cycle. In this work, we show that Mcm2, Mcm3, and Mcm5 self-interact and interact with one another to form complexes. Mcm2 and Mcm3 are abundant proteins, present in approximately 4 X 10(4) and 2 X 10(5) copies per cell, respectively. Reducing the dosage of Mcm2 by half results in diminished usage of specific replication origins. These results together suggest that a significant molar excess of Mcm proteins relative to replication origins is required for the proper initiation of all replication origins.

Cell Cycle Proteins↗
PubMed1995

The yeast Mcm1 protein is regulated posttranscriptionally by the flux of glycolysis.

Mcm1 is a multifunctional protein which plays a role both in the initiation of DNA replication and in the transcriptional regulation of diverse genes in Saccharomyces cerevisiae. The mcm1-1 mutation results in instability of minichromosomes and alpha-specific sterility. Second-site suppressors that restore minichromosome stability but not fertility to the mcm1-1 mutant were isolated. Two of the suppressors, pgm1-1 and pgm1-2, are mutant alleles of PGM1 which encodes a glycolytic enzyme, phosphoglycerate mutase. We show that the pgm1-1 mutation suppresses the minichromosome maintenance (Mcm) defect by increasing the protein activity or level of Mcm1-1 posttranscriptionally. This increase in the intracellular Mcm1-1 activity is sufficient to suppress the Mcm defect but only minimally suppresses the mating defect. Mutations in genes encoding other glycolytic enzymes, such as eno2::URA3, can also suppress the Mcm phenotype of mcm1-1. Suppression by these glycolytic enzyme mutations correlates with a reduced rate of glycolysis rather than a reduced rate of cell growth. This study suggests that in response to changes in their nutritional states yeast cells may attain homeostasis by modulating the activity of global regulators like Mcm1, which plays a central role in the regulation of energy-expensive anabolic processes.

Alleles↗
PubMed1994

The MCM2-3-5 proteins: are they replication licensing factors?

DNA replication occurs only once in each normal mitotic cell cycle. To explain this strict control, a 'licensing factor' was proposed to enter the nucleus periodically as the nuclear envelope disintegrates and reassembles at the end of mitosis. Inactivation of licensing factor immediately following initiation of DNA synthesis would prevent reinitiation until after the next mitosis. The MCM2-3-5 proteins of Saccharomyces cerevisiae may be yeast's equivalent of licensing factor: they are present in the nucleus only between M and S phase, bind to chromatin and are important for the initiation of DNA replication.

Journal Article↗
PubMed1993

Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA.

We have identified a nuclease activity that is specific for G4 tetrastranded DNA. This activity, found in a partially purified fraction for a yeast telomere-binding protein, binds to DNA molecules with G4 tetrastranded structure, regardless of their nucleotide sequences, and cleaves the DNA in a neighboring single-stranded region 5' to the G4 structure. Competition with various G4-DNA molecules inhibits the cleavage reaction, suggesting that this nuclease activity is specific for G4 tetrastranded DNA. The existence of this enzymatic activity that reacts with G4 DNAs but not with single-stranded or Watson-Crick duplex DNAs suggests that tetrastranded DNA may have a distinct biological function in vivo.

Base Sequence↗
PubMed1993

Cell cycle-regulated nuclear localization of MCM2 and MCM3, which are required for the initiation of DNA synthesis at chromosomal replication origins in yeast.

MCM2 and MCM3 are two genetically interacting and structurally related proteins essential for growth in Saccharomyces cerevisiae. Mutants defective in these proteins affect the stability of minichromosomes in general, but the severity of the defect is dependent on the autonomously replicating sequence (ARS) that drives the replication of that plasmid. In this paper we show by two-dimensional gel electrophoresis that the initiation of DNA synthesis at chromosomal replication origins is also reduced in frequency in these mutants. We show further that the nuclear and subnuclear localizations of the MCM2 and MCM3 proteins are temporally regulated with respect to the cell cycle. These proteins enter the nucleus at the end of mitosis, persist there throughout G1 phase, and disappear from it at the beginning of S phase. Once inside the nucleus, a fraction of the MCM2 and MCM3 proteins becomes tightly associated with DNA. The association of MCM2 and MCM3 with chromatin presumably leads to the initiation of DNA synthesis, and their subsequent disappearance from the nucleus presumably prevents reinitiation of DNA synthesis at replication origins. This temporally and spatially restricted localization of MCM2 and MCM3 in the nucleus may serve to ensure that DNA replication occurs once and only once per cell cycle.

Cell Cycle↗
PubMed1992

CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences.

Saccharomyces cerevisiae cells containing mutations in the cell-division-cycle gene CDC46 arrest with a large bud and a single nucleus with unreplicated DNA at the non-permissive temperature. This G1/S arrest, together with the increased rates of mitotic chromosome loss and recombination phenotype, suggests that these mutants are defective in DNA replication. The subcellular localization of the CDC46 protein changes with the cell cycle; it is nuclear between the end of M phase and the G1/S transition but is cytoplasmic in other phases of the cell cycle. Here we show that CDC46 is identical to MCM5, based on complementation analysis of the mcm5-1 and cdc46-1 alleles, complementation of the minichromosome maintenance defect of mcm5-1 by CDC46, and the genetic linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 also show a minichromosome maintenance defect thought to be associated with DNA replication initiation at autonomously replicating sequences. Taken together, these observations suggest that CDC46/MCM5 acts during a very narrow window at the G1/S transition or the beginning of S phase by virtue of its nuclear localization to effect the initiation of DNA replication at autonomously replicating sequences.

Amino Acid Sequence↗
PubMed1992

Chromosome loss, hyperrecombination, and cell cycle arrest in a yeast mcm1 mutant.

The original mcm1-1 mutant was identified by its inability to propagate minichromosomes in an ARS-specific manner, suggesting that it is defective in the initiation of DNA synthesis at ARSs. This mutant is also defective in expression of alpha-mating-type-specific genes. Further genetic and biochemical studies confirmed that Mcm1 is a transcription factor that mediates the transcriptional regulation of a number of genes, including genes outside of the mating type complement, by interacting with different cofactors. Although MCM1 is an essential gene, none of the previously characterized mcm1 mutants exhibits significant growth defects. To assess which of the many roles of Mcm1 is essential for growth, we constructed and characterized a temperature-sensitive conditional mutant of mcm1, mcm1-110L. This mutant exhibits a temperature-dependent cell-cycle arrest, with a large, elongated bud and a single, undivided nucleus that has a DNA content of close to 2n. In addition, it shows elevated levels of chromosome loss and recombination. In spite of the severity of the mcm1-110L mutation, this mutant still retains an ARS-specific pattern of minichromosome instability. All of these phenotypes are precisely those exhibited by mutants in three MCM genes, MCM2, MCM3, and MCM5/CDC46, that have been shown to play interacting roles in the early steps of DNA replication.

Cell Cycle↗