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Replicating Sequences (Minchromosome Instability/Cell Cyle-Dependent Nuclear Lctlon/Protein Family Involved in DNA Replication) YANRU CHEN*, KEVIN M

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Replicating Sequences (Minchromosome Instability/Cell Cyle-Dependent Nuclear Lctlon/Protein Family Involved in DNA Replication) YANRU CHEN*, KEVIN M Proc. Nati. Acad. Sci. USA Vol. 89, pp. 10459-10463, November 1992 Biochemistry CDC46/MCM5, a yeast protein whose subcellular localization is cell cycle-regulated, is involved in DNA replication at autonomously replicating sequences (minchromosome instability/cell cyle-dependent nuclear lctlon/protein family Involved in DNA replication) YANRU CHEN*, KEVIN M. HENNESSYt, DAVID BOTSTEINt, AND BIK-KWOON TYE* *Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853; and tDepartment of Genetics, Stanford University, Stanford, CA 94305 Contributed by David Botstein, July 31, 1992 ABSTRACT Saccharomyces cerevisiae cells containing mu- With this approach, a number of minichromosome main- tations in the cell-division-cycle gene CDC46 arrest with a large tenance mutants were isolated. Mutants in several comple- bud and a single nucleus with unreplicated DNA at the non- mentation groups were of particular interest because they permissive temperature. This G1/S arrest, together with the show an ARS-specific minichromosome maintenance defect. increased rates of mitotic chromosome loss and recombination These mutants have an effect on the stability of all minichro- phenotype, suggests that these mutants are defective in DNA mosomes tested, but those carrying certain ARSs are much replication. The subcellular localization of the CDC46 protein more affected than others (3). Although minichromosome changes with the cell cycle; it is nuclear between the end of M instability seems to be a general property of replication- phase and the G1/S transition but is cytoplasmic in other defective mutants, this ARS specificity is unique to the mcm phases of the cell cycle. Here we show that CDC46 is identical mutants, suggesting that they harbor defects in DNA repli- to MCM5, based on complementation analysis of the mcm5-1 cation targeted at ARSs (4). Three of these MCM genes, and cdc46-1 alleles, complementation of the minichromosome MCMI, MCM2, and MCM3, have been cloned and further maintenance defect of mcm5-1 by CDC46, and the genetic characterized. Mutants in MCMJ (4), MCM2 (5), and MCM3 linkage of these two genes. Like mcm5-1, cdc46-1 and cdc46-5 (6) all show increased chromosome loss and recombination, also show a mimchromosome maintenance defect thought to be characteristic of mutants defective in DNA replication (7). associated with DNA replication initiation at autonomously Furthermore, instability of minichromosomes in the mcm2-1 replicating sequences. Taken together, these observations sug- mutant has been shown to be due to plasmid loss or nonrep- gest that CDC46/MCM5 acts during a very narrow window at lication rather than missegregation (5). the G,/S transition or the inning of S phase by virtue of its MCMJ encodes a DNA-binding protein that acts both as a nuclear localization to effect the initiation of DNA replication transcription factor (8, 9) and a replication initiator (10) by at autonomously replicating sequences. binding to regulatory sequences upstream of a number of mating type-specific genes (11-13) and to ARSs (C. Christ, V. The eukaryotic genome is replicated once and only once Chang, and B.-K.T., unpublished results). Genetic analysis within a defined period ofthe cell cycle. Replication requires indicates that MCM2 and MCM3 carry out related functions the coordinated initiation ofDNA synthesis at multiple sites. (14). Double mutants ofmcm2 and mcm3 result in a synthetic These initiation events are under cell cycle control such that lethal phenotype. Furthermore, overproduction of MCM2 DNA synthesis occurs only during S phase and reinitiation is complements the mcm3-J mutation while overproduction of prevented until the next cell cycle. Molecular events that lead MCM3 is lethal to the mcm2-1 mutant. Interestingly, MCM2 to and MCM3 show striking similarity in their protein sequences replication initiations in response to cell cycle signals are (14). This homology extends to other known proteins, CDC46 largely unknown. of S. cerevisiae and CDC21 of Schizosaccharomyces pombe The genome of the budding yeast Saccharomyces cerevi- (A. Coxon and S. Kearsey, personal communication). Phe- siae is estimated to contain 200-400 replication origins that notypes of cdc46 and Cdc21- mutant strains are consistent can be cloned on plasmids as autonomously replicating with defects in DNA replication. This finding led us to sequences (ARSs) (1, 2). The cloning of ARSs on plasmids postulate that these proteins constitute a family of at least provides a convenient means to identify gene products in- four members, all of which play important roles in the early volved in ARS function by screening for mutants that desta- steps of DNA replication (14, 15). bilize minichromosomes containing one of these ARSs (3). Conditional mutations that completely block the initiation The most attractive feature of this approach is that it allows step of all DNA synthesis are expected to have a Gl-arrest one to isolate viable mutants with a defect in a specific phenotype. The cdc46 mutations were originally identified as function without relying on general phenotypes, such as cell allele-specific suppressors ofcdc45 and cdc54 mutations (16). cycle arrest, which often give no information on the execu- These cdc mutants arrest at the G1/S transition or the tion point of the lesion. This feature is particularly important beginning of the S phase of the cell cycle. The subcellular if replication origins are coordinately regulated in groups via localization of CDC46 has been shown to be under cell cycle shared initiator proteins. Thus, elimination of a particular control: it appears in the nucleus at late M phase and persists initiator protein that is responsible for the initiation of only a in the nucleus until the G1/S transition (17). In addition to the subset ofARSs may not necessarily be lethal to the cell since striking homology of CDC46 to the MCM2 and MCM3 replication of inactive replicons by merging neighboring proteins, the cdc46-1 mutant exhibits phenotypes similar to replicons would be expected. those of the mcm2 and mcm3 mutants, showing high rates of mitotic chromosome loss and recombination (15). If MCM2, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: ARS, autonomously replicating sequence; FOA, in accordance with 18 U.S.C. §1734 solely to indicate this fact. 5-fluoroorotic acid; Cm-ura, complete medium lacking urocil. 10459 10460 Biochemistry: Chen et al. Proc. Nat!. Acad. Sci. USA 89 (1992) MCM3, and CDC46 are members of a protein family related fluoroorotic acid) plates were made according to Boeke et al. in structure and function, it seems likely that cdc46 mutants (22). should also show an ARS-specific minichromosome mainte- Construction of Heterozygous Diploids from Haploids with nance defect. In fact, we surmised that CDC46 may corre- Simfiar Genetic Markers. To cross strains, such as RM11-2A spond to one of the original ARS-specific MCM genes and 8534-8C, that contain many of the same selectable identified by Maine et al. (3). markers, 8534-8C was transformed with YCp1O1 (carrying In this paper, we show that CDC46 is identical to MCMS. LEU2). Diploids were selected on complete medium lacking We further present data to show that CDC46 is required for histidine and leucine (Cm-his-leu). Diploids were con- minichromosome maintenance in an ARS-specific manner. firmed by their ability to sporulate and their inability to mate. Genetic Techniques and DNA Transformation. Mating, MATERIALS AND METHODS sporulation, and meiotic tetrad analysis were carried out as Strains and Plasmids. Escherichia coli strain DH5a (BRL) described (21). Transformation of yeast cells with plasmids was used for plasmid construction and amplification. Yeast was carried out by a modified lithium acetate method (23). strain 8534-8C (MATa leu2-3,112 ura3-52 his4-A34) is the Mitotic Stability Assay for Plsmids. The stability assay parent strain of the mcm mutants (3, 18). The mcmS-1 strain measured the cumulative loss of minichromosomes from a is strain 1-11 described by Maine et al. (3). Two strains of yeast culture after 10-12 generations of nonselective growth mcmS-1 that resulted from backcross were used in this study: at specified temperatures (3). Complete medium lacking RM11-2A (MATa leu2-3,112 ura3-52 mcmS-1) and RM11-2B uracil (Cm-ura) served as the selective medium for retaining (MATa leu2-3,112 his4-A34 mcmS-1). R61-2B (MATa, leu2-3, YCp5OCDC46 and YCp5O in the cells, while Cm-ura-leu 112 ura3-S2 his3-11,lS mcm3-1) has been described (6). The additionally selected for the test plasmid. The final stability double mutant mcm2-1 mcm4-1 (MATa leu2-3,112 ura3-52 (F) of the test plasmid was calculated as the percentage of his4A34 mcm2 mcm4) was constructed by H. Yan (Cornell cells growing on Cm-ura-leu after 10-12 generations of University, Ithaca, NY). DBY2028 (MATa ura3-52 ade2-1 nonselective growth on Cm-ura plates. The minichromo- lys2-801 leu2-3,112 cdc46-1) and YRC1 (MATa lys2-801 some loss rate is defined as the rate of plasmid loss per cell ade2-101 his3-A200 leu2-Al ura3-52 cdc46-5) have two dif- division. It is expressed as 1-(F/l)1/N, where I is the initial ferent cdc46 alleles that suppress cdc45-1 and cdc54-1, re- percentage of plasmid-containing cells and F is the percent- spectively (17). The strain YPH500 (MATa ura3-52 lys2-801 age of plasmid-containing cells after N generations of non- ade2-101 trpl-A63 his3A200 leu2-AJ) used for genetic map- selective growth. ping was obtained from the Yeast Genetic Stock Center (Berkeley, CA). RESULTS The plasmids used in this study are listed in Table 1. CDC46 Complements mcmS but Not mcm2, mcm3, mcm4, or YCp5OCDC46 has a 15.6-kilobase (kb) fragment containing the mcm7.
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