Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase

Aparicio, Oscar M.; Weinstein, Deborah M; Bell, Stephen P.

doi:10.1016/s0092-8674(01)80009-x

Cited by 693 publications

(845 citation statements)

References 48 publications

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“…As cells progressed into S-phase the association of Mcm2 with the origin decreased and the association of Mcm2p with origin-distal DNA fragments increased. This pattern implies association with the replication fork, as has been previously described (Aparicio et al, 1997;Tanaka et al, 1997). We found that when TetO 7 -SMC4 was shut off, Mcm2p binding to ARS1 decreased as cells progressed into S phase, but Mcm2p failed to then associate with origin-distal regions.…”

Section: S Phasesupporting

confidence: 84%

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Peña‐Castillo

Mnaimneh

et al. 2006

MBoC

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Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO 7 promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication. INTRODUCTIONCell division is a fundamental biological process that in all organisms consists of a series of closely coordinated events. In the budding yeast Saccharomyces cerevisiae, the cell division cycle begins with an initial growth phase, G1, during which time the cell increases in mass and volume. The transition from G1 into S phase is marked by progression through Start (the point of commitment to cell division), initiation of nuclear DNA synthesis, and the emergence of a bud that will form the new daughter cell. S phase is followed by a second growth phase, G2, which is in turn followed by nuclear division, and then cell separation. A very similar series of events occurs in all eukaryotes, with the obvious exception of bud emergence. Because of the fundamental biological importance of cell cycle progression and its importance in both human development and diseases such as cancer, identification of the molecular determinants of specific stages of the eukaryotic cell cycle has been a subject of intense study for several decades.The morphological landmarks of the cell cycle stages in budding yeast, most notably the size of the bud relative to the size of the mother cell, allows the identification of mutants blocked at specific stages of the cell cycle and thereby forms the basis for the classic cell cycle screens (Hartwell et al., 1970;Culotti and Hartwell, 1971;Hartwell, 1971aHartwell, , 1971bHartwell, , 1973Moir et al., 1982). These genetic screens, using conditional temperature-sensitive mutants, identified more than 50 genes that are required for specific stages in the cell division cycle and so were termed CDC genes. On average 4.6 alleles were identified for each CDC gene in the original screens, suggesting the number of cdc mutan...

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Section: S Phasesupporting

confidence: 84%

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Peña‐Castillo

Mnaimneh

et al. 2006

MBoC

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“…Orc1 protein was tagged by 3ϫ HA epitope tag cassette at its C terminus using the plasmid p306ORC1-HA/C (18). An ⌬ume6 mutant is a deletion mutant that is constructed by insertion of the KanMX cassette (19).…”

Section: Methodsmentioning

confidence: 99%

Perturbation of the Activity of Replication Origin by Meiosis-specific Transcription

Mori

Shirahige

2007

Journal of Biological Chemistry

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We have determined the activity of all ARSs on the Saccharomyces cerevisiae chromosome VI as chromosomal replication origins in premeiotic S-phase by neutral/neutral two-dimensional gel electrophoresis. The comparison of origin activity of each origin in mitotic and premeiotic S-phase showed that one of the most efficient origins in mitotic S-phase, ARS605, was completely inhibited in premeiotic S-phase. ARS605 is located within the open reading frame of MSH4 gene that is transcribed specifically during an early stage of meiosis. Systematic analysis of relationships between MSH4 transcription and ARS605 origin activity revealed that transcription of MSH4 inhibited the ARS605 origin activity by removing origin recognition complex from ARS605. Deletion of UME6, a transcription factor responsible for repressing MSH4 during mitotic S-phase, resulted in inactivation of ARS605 in mitosis. Our finding is the first demonstration that the transcriptional regulation on the replication origin activity is related to changes in cell physiology. These results may provide insights into changes in replication origin activity in embryonic cell cycle during early developmental stages.Eukaryotic chromosomes consist of multiple replication units. Each origin of DNA replication is strictly controlled to fire only once in the cell cycle in the fixed sequential order (1, 2). This temporal program for origin firing is utilized for the maintenance of genome integrity through DNA replication checkpoint mechanism (3, 4). It was shown that a checkpoint effector kinase Rad53 repressed firing of late origins when replication was perturbed by hydroxy urea or methyl methane sulfonate. Furthermore, recent genome-wide studies of eukaryotic chromosomal DNA replication using Saccharomyces cerevisiae have provided us with a kinetic map of progression of DNA replication along the chromosome (5, 6). However, these studies were restricted to DNA replication during mitotic cell cycle and little is known about how these multiple replication origins behave under different cell cycle, i.e. meiotic cell cycle.The decision to enter the meiotic cell cycle is made in G 1 phase and this affects the way in which the G 1 /S transition is controlled. In budding yeast (S. cerevisiae), poor nutrient conditions are the cue to embark on the meiotic cell cycle, which culminates in the production of spores (7). The replication of chromosome is the first detectable cytological event in meiosis. The coordinated synthesis of genomic DNA requires multiple levels of regulation and a large number of gene products. Genetic analysis in S. cerevisiae indicates that the replicative machinery used to synthesize DNA in vegetative cells is also required for the duplication of chromosome in meiosis (8 -10).In S. cerevisiae, mitotic S-phase and premeiotic S-phase appear to be differently regulated. For example, premeiotic S-phase is 1.5-2 times longer than mitotic S-phase (11). However, replication kinetics as measured by neutral/neutral twodimensional gel electrophoresis of 100-kb s...

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“…In the yeast Saccharomyces cerevisiae, but also in many other organisms, the binding of Cdc6 to the origin recognition complex (ORC) is a critical step in the formation of pre-RCs (Liang et al, 1995;Cocker et al, 1996;Detweiler and Li, 1997) and essential for subsequent loading of Mcm proteins (Mcm2-7; Piatti et al, 1996;Santocanale and Diffley, 1996;Aparicio et al, 1997;Donovan et al, 1997;Tanaka et al, 1997). After licensing the origin by loading Mcms, endogenous Cdc6 dissociates from the replicative complex and only reassociates with chromatin late in M-phase Weinreich et al, 1999) when cyclin-dependent protein kinase (Clb-Cdc28) activities are absent.…”

Section: Introductionmentioning

confidence: 99%

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

Honey

Futcher

2007

MBoC

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The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of these mechanisms individually. We find that when Cdc6 is forced into the nucleus during late G1 or S, it will not substantially reload onto chromatin no matter whether its CDK sites are present or not. In contrast, at a G2/M nocodazole arrest, Cdc6 will reload onto chromatin if and only if its CDK sites have been removed. Trace amounts of nonphosphorylatable Cdc6 are dominant lethal in strains bearing nonphosphorylatable Orc2 and Orc6, apparently because of rereplication. This synthetic dominant lethality occurs even in strains with wild-type MCM genes. Nonphosphorylatable Cdc6, or Orc2 and Orc6, sensitize cells to rereplication caused by overexpression of various replication initiation proteins such as Dpb11 and Sld2.

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Components and Dynamics of DNA Replication Complexes in S. cerevisiae: Redistribution of MCM Proteins and Cdc45p during S Phase

Cited by 693 publications

References 48 publications

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

A Survey of Essential Gene Function in the Yeast Cell Division Cycle

Perturbation of the Activity of Replication Origin by Meiosis-specific Transcription

Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

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