The Role of Nucleotide Binding and Hydrolysis in the Function of the Fission Yeast cdc18⁺ Gene Product

Corresponding author: G. Steven Martin, Division of Biochemistry and Molecular Biology, Department of Molecular and Cell Biology, University of California, 401 Barker Hall #3204, Berkeley, CA 94720-3204., smartin{at}socrates.berkeley.edu (E-mail)

Communicating editor: P. G. YOUNG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The fission yeast cdc18⁺ gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18⁺ gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C–2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

IN eukaryotes, DNA replication is initiated at many origin sequences within a single cell (CAIRNS 1966; HUBERMAN and RIGGS 1968 ). To maintain genome integrity, initiation must occur at each origin once and only once per round of replication, and DNA replication and mitosis must be alternating events. The currently favored model for initiation of DNA replication proposes a two-step process in which the two steps are temporally distinct and mutually exclusive (reviewed in MUZI-FALCONI et al. 1996B ). First, in late M or early G₁ phases, a "licensing factor" binds to or otherwise modifies origins of replication, making them competent for initiation. Then, after passage through START, an S-phase-promoting factor is activated and replication is initiated at licensed origins. As each origin fires, it is converted to an unlicensed state. Because licensing is restricted to late M/early G₁, origins cannot be relicensed and DNA cannot be rereplicated without an intervening mitosis.

An understanding of the biochemical events that result in origin licensing has been facilitated by the definition of origin sequences in Saccharomyces cerevisiae and the identification of proteins that interact with these sequences. A complex of six polypeptides known as the origin recognition complex (ORC) is bound to origin sequences throughout the cell cycle (BELL and STILLMAN 1992 ; DIFFLEY et al. 1994 ). Homologs of several of the ORC proteins have been identified in Schizosaccharomyces pombe, plants, and metazoans (GAVIN et al. 1995 ; GOSSEN et al. 1995 ; MUZI-FALCONI and KELLY 1995 ; CARPENTER et al. 1996 ; GRALLERT and NURSE 1996 ; LEATHERWOOD et al. 1996 ; ROWLES et al. 1996 ; TAKAHARA et al. 1996 ). Upon digestion with DNase, the binding of ORC to origin sequences produces a characteristic genomic footprint that can be reproduced in vitro using purified ORC components (BELL and STILLMAN 1992 ; DIFFLEY and COCKER 1992 ). In late mitosis, this footprint is extended and an additional region of protection remains visible throughout G₁ until S phase begins (DIFFLEY et al. 1994 ). The appearance of this extended footprint is coincident with the period of the cell cycle when the DNA is licensed; it has been proposed that the extension of the footprint represents licensing factor joining ORC.

Biochemical and genetic evidence suggests that the licensing reaction is initiated by a conserved nuclear protein, the product of the CDC6 gene in S. cerevisiae and the cdc18⁺ gene in S. pombe. In S. cerevisiae, the cdc6-1 mutant exhibits a decrease in the frequency of origin firing (LIANG et al. 1995 ). This phenotype appears to be specific for genes whose products function in initiation, including the origin recognition proteins Orc2 and Orc5 (FOX et al. 1995 ; LIANG et al. 1995 ). In S. pombe, the cdc18⁺ gene is essential for DNA replication and also plays a role in the checkpoint that prevents mitosis in the absence of DNA replication (KELLY et al. 1993 ). Overexpression of Cdc18 to very high levels disrupts alternation of licensing and mitosis, causing cells to undergo repeated rounds of DNA replication without intervening mitoses (NISHITANI and NURSE 1995 ; MUZI-FALCONI et al. 1996A ). Both Cdc6 and Cdc18 interact with the ORC/Orp complex (GRALLERT and NURSE 1996 ; LEATHERWOOD et al. 1996 ), and experiments using Xenopus egg extracts have demonstrated that XCdc6 associates with chromatin in an ORC-dependent manner (COLEMAN et al. 1996 ). Finally, CDC6 is required for both formation and maintenance of the prereplicative footprint (COCKER et al. 1996 ), suggesting that Cdc6 itself may function as a component of the licensing factor or be required for licensing to occur.

The biochemical function of Cdc6/Cdc18 has not been precisely defined. Several lines of evidence suggest that Cdc6/Cdc18 functions to recruit a multisubunit complex containing the minichromosome maintenance (MCM) proteins to prereplicative chromatin. The MCM genes were first identified in S. cerevisiae, where they are required for the replication and maintenance of multi-copy plasmids (MAINE et al. 1984 ; SINHA et al. 1986 ) and for chromosomal replication (HENNESSY et al. 1990 ; YAN et al. 1993 ). Biochemical assays for licensing activity in Xenopus egg extracts have been developed and used to purify licensing-factor-containing fractions from S-phase egg extracts (CHONG et al. 1995 ; KUBOTA et al. 1995 ). These fractions were found to contain a complex of proteins homologous to the MCM proteins of S. cerevisiae. Immunodepletion experiments have confirmed that the Xenopus MCM proteins are essential for the licensing reaction (MADINE et al. 1995 ). In further studies, the immunodepletion of XCdc6 prevented association of XMcm3 with chromatin (COLEMAN et al. 1996 ). It has also been shown that Cdc6 is required for the association of S. cerevisiae MCM proteins with chromatin (APARICIO et al. 1997 ; DONOVAN et al. 1997 ; TANAKA et al. 1997 ); however, once MCMs are bound, Cdc6 can be removed from the chromatin without affecting the association of MCM-containing complexes with chromatin (DONOVAN et al. 1997 ). Finally, Cdc18 and Cdc21 (an S. pombe MCM protein) both coprecipitate with Orp1, suggesting that these three proteins form a complex in vivo (GRALLERT and NURSE 1996 ).

Examination of the sequences of Cdc18 and its homologs from S. cerevisiae, Xenopus, and human has revealed several recognizable sequence motifs that are well conserved. All of the homologs contain both the Walker A and B box sequences commonly found in proteins that bind and hydrolyze nucleotides (WALKER et al. 1982 ). To assess the importance of these motifs for Cdc18 function, we have examined the effects of mutations in these sequences on cell-cycle progression. We have found that both the A and B box sequences are required for Cdc18 function, indicating that nucleotide binding and hydrolysis by Cdc18 are both essential. Notably, mutants carrying disruptions of the A and B box sequences display different phenotypes, and the phenotype displayed by the A box mutant is dependent on cell-cycle position at the time that wild-type cdc18⁺ expression is repressed. These observations suggest that nucleotide binding and hydrolysis by Cdc18 are required for distinct aspects of Cdc18 function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and methods:
Strains were constructed using standard genetic techniques as previously described (MORENO et al. 1991 ). The cdc18::ura4⁺, leu1-32, ade6-M210/ade6-M216, ura4-D18, his3-D1 strain was derived from the previously described cdc18 strain (KELLY et al. 1993 ) and a his3-D1, ura4-D18, leu1-32, ade6-M216, h⁺ strain (BURKE and GOULD 1994 ). All cells were grown in minimal medium (EMM) + supplements as described (MORENO et al. 1991 ). Thiamine was added to a final concentration of 20 µg/ml to repress expression from the nmt1⁺ promoter. Hydroxyurea was added to a final concentration of 12 mM and was removed by washing the cells three times in cold medium. The cdc25-22 strains were grown at a permissive temperature of 25° and were arrested at a restrictive temperature of 36°.

Plasmids:
The plasmid pBM001 contains a copy of cdc18⁺ under the control of its own promoter and a mutant leu1 allele. pBM001 was derived from pJK148 (KEENEY and BOEKE 1994 ) in several steps. The 100-bp NruI-NdeI fragment of pJK148 was removed to generate pDAD70, which contains a mutationally inactivated leu1 gene. This allows homologous integration events to be selected because in a leu1-32 strain, recombination between the chromosomal and plasmid-borne copies of leu1 is required for a functional copy of the leu1⁺ gene to be generated. A wild-type copy of cdc18⁺ was introduced within a 3-kb EaeI-EcoRV genomic fragment (KELLY et al. 1993 ) at the HincII and NotI sites of pDAD70 to generate pDAD74. A 719-bp ClaI-ApaI fragment containing part of the multiple cloning site was removed from the vector backbone of pDAD74 to generate pDAD76. Finally, three copies of the middle T antigen epitope EYMPME were introduced at the C terminus of cdc18⁺ by PCR to generate pBM001.

pDAD112 was constructed by excision of the LEU2 gene from pREP81:cdc18⁺ (KELLY et al. 1993 ) by digestion with HindIII and insertion by blunt-end ligation of a 1.75-kb DraI-EcoRV fragment from pAF1 (BURKE and GOULD 1994 ) that contained the his3⁺ gene.

Construction of cdc18 mutant strains:
Mutant alleles of cdc18⁺ were constructed by in vitro oligonucleotide-directed mutagenesis using sequential PCR steps (CORMACK 1991 ). The sequences of the mutagenic oligonucleotide pairs are 5'agt ggt gcc cct ggc aca gga gct acc gtt ctg ctt3'/5'aag cag aac ggt agc tcc tgt gcc agg ggc acc act3' for the cdc18-K205A allele and 5'atc cat tgc agc tag cac aat aat gac3'/5'att att gtg cta gct gca atg gat cac 3' for the cdc18-DE286AA allele. The mutant alleles were introduced at the leu1 locus of a cdc18::ura4⁺, leu1-32, ade6-M210/ade6-M216, ura4-D18, his3-D1 strain by homologous integration as previously described (KEENEY and BOEKE 1994 ). pBM001 was used as the integration vector and pDAD70 as an empty vector control. Single-copy integrants were confirmed by Southern blot. The diploid integrants were transformed with pDAD112. Diploids were sporulated in EMM containing 5 g/liter glutamate as a limiting nitrogen source and vegetative cells were removed by digestion with helix pomatia juice (Sepracor, Marlborough, MA) as previously described (MAIORANO et al. 1996 ). cdc18::ura4⁺, leu1⁺::cdc18-X, ade6-M210 or M216, ura4-D18, his3-D1 haploids carrying the pDAD112 plasmid were auxotrophically selected.

Flow cytometry and 4',6'-diamidino-2-phenylindole dihydrochloride staining:
Cells were ethanol fixed and analyzed by flow cytometry as described (SAZER and SHERWOOD 1990 ), except that DNA was stained with 1 µM Sytox Green (Molecular Probes, Eugene, OR). Flow cytometry was performed using a Coulter (Hialeah, FL) Epics XL-MCL flow cytometer and System II software. Septation indices were determined by microscopic analysis of 200 cells per sample. Fixed cells were rehydrated in water, heat fixed to slides at 70°, and stained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) mounting solution (1 µg/ml DAPI, 1 mg/ml p-phenylenediamine, 50% glycerol).

Pulsed-field gel electrophoresis:
Chromosomal DNAs were prepared and electrophoresis was carried out as previously described (KELLY et al. 1993 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Nucleotide-binding and hydrolysis motifs are required for Cdc18 function:
To determine whether nucleotide binding and hydrolysis are required for Cdc18 function, we introduced mutations in the Walker A and B box motifs of cdc18⁺ (Figure 1A). These two sequence motifs are highly conserved in purine nucleotide-binding proteins (WALKER et al. 1982 ). The A box has the consensus sequence GXXGXGKT, and the lysine residue of this motif is invariant in all nucleotide-binding proteins. In many proteins, mutation of this lysine has been shown to disrupt nucleotide binding and/or biochemical function (BOOHER and BEACH 1986 ; ROZEN et al. 1989 ; SONG et al. 1990 ; HABER and WALKER 1991 ; PAUSE and SONENBERG 1992 ; ZHU and WELLER 1992 ; LIM et al. 1993 ; KLEMM et al. 1997 ). The B box consensus Hy-Hy-Hy-Hy-DEXD/H (where Hy is a hydrophobic residue) is found in many proteins involved in DNA replication and/or recombination (HODGMAN 1988 ; GORBALENYA et al. 1989 ; KOONIN 1993 ). Mutation of the aspartate and/or glutamate residues of this motif has been shown to disrupt the biochemical activities of several proteins (SONG et al. 1990 ; SCHMID and LINDER 1991 ; ZHU and WELLER 1992 ) and specifically disrupts ATP hydrolysis in at least three cases (PAUSE and SONENBERG 1992 ; BROSH and MATSON 1995 ; WENG et al. 1996 ). The mutations that were introduced into cdc18⁺ were K205A in the A box and DE286AA in the B box.

View larger version (43K): In this window In a new window Download PPT slide   Figure 1. Nucleotide-binding and hydrolysis motifs are required for cdc18+ function. (a) A schematic diagram of the Cdc18 protein. The region of homology with Orc1 is indicated by the solid box. The hatched regions show the locations of nucleotide-binding (amino acids 200–207) and hydrolysis (amino acids 286–289) motifs. The sequences of these motifs from Cdc18 and its homologs are shown. Mutant alleles in which these motifs are disrupted were constructed by replacing the underlined residues with alanine (K205A and DE286AA). (b) Haploid strains were constructed carrying a deletion of the cdc18 open reading frame at the cdc18 locus, a plasmid-borne thiamine-repressible wild-type copy of cdc18+, and a mutant allele of cdc18 integrated at the leu1 locus. Control strains carrying wild-type cdc18+ or empty vector at the leu1 locus were also constructed. Each strain was streaked on media with and without thiamine at 30°. Viability was scored after 3 days.

To characterize the phenotypes associated with the K205A and DE286AA mutations, a single copy of each of the mutant alleles was introduced into the genome of a cdc18 strain by integration at the leu1 locus. Wild-type cdc18⁺ was provided on a plasmid under control of a modified form of the thiamine-repressible nmt1⁺ promoter to allow for recovery of nonfunctional mutant integrants. Equivalent strains with wild-type cdc18⁺ or empty vector integrated at the leu1 locus were also constructed for use as positive and negative controls, respectively. Each of the strains was then plated on medium with and without thiamine. In the presence of thiamine, expression of wild-type cdc18⁺ from the nmt1 promoter is repressed, making the cell dependent on the function of the mutant protein for viability. Both the K205A and DE286AA mutant strains are inviable in the presence of thiamine (Figure 1B). Immunoblotting analysis indicated that the mutant proteins are expressed at the same level as the wild-type Cdc18 protein (data not shown). These data indicate that nucleotide binding and hydrolysis are both required for Cdc18 function.

Nucleotide binding and hydrolysis mutants display distinct phenotypes:
The cdc18⁺ gene is required for initiation of DNA replication and for mitotic checkpoint control. To determine the effect of the K205A and DE286AA mutations on both DNA replication and mitotic checkpoint, the terminal phenotypes of the mutant strains described above were further characterized. Mutant strains were grown in minimal medium and thiamine was added to the cultures to suppress expression of the plasmid-borne copy of wild-type cdc18⁺. This is expected to result in rapid depletion of the cellular pool of wild-type Cdc18, as the protein has a half-life of <5 min (MUZI-FALCONI et al. 1996A ). Cells were fixed at intervals after the addition of thiamine. To assess the replicative function of the mutant alleles, fixed cells were stained with Sytox Green and examined by flow cytometry to determine DNA content. In addition, fixed cells were stained with DAPI and observed using a fluorescence microscope to detect abnormal nuclear morphology, which is indicative of loss of the checkpoint function. Finally, the percentage of cells displaying a septum (the septation index) was calculated to determine whether the cells continue to undergo cell division after the addition of thiamine.

As expected, the wild-type control strain was unaffected by the addition of thiamine (Figure 2). The DNA content of the wild-type population remained at 2C throughout the course of the experiment and the septation index did not vary significantly. In contrast, the empty vector control displayed the null phenotype previously described (KELLY et al. 1993 ; Figure 2). By 3 hr after the addition of thiamine, most of the population had a 1C DNA content due to a defect in initiation of DNA replication in the absence of Cdc18. A small fraction of cells retained a >1C DNA content, possibly due to persistence of wild-type Cdc18 protein or residual cdc18⁺ expression in the presence of thiamine. At later times, the peak continued to shift to the left as cells with a <1C DNA content accumulated, confirming that the checkpoint that functions to inhibit mitosis in the absence of DNA replication is also disrupted in the absence of Cdc18. Consistent with this interpretation, the septation index decreased throughout the course of the experiment, but never reached zero, indicating that the cells were continuing to cycle. In addition, DAPI staining revealed that at 7 hr after the addition of thiamine, 50% of the cells exhibited the abnormal nuclear morphology that is often associated with checkpoint defects (data not shown), including "cut" cells, anucleate cells, and cells that had asymmetrically segregated their DNA.

View larger version (26K): In this window In a new window Download PPT slide   Figure 2. DNA content and septation indices of cdc18 mutants. The haploid strains described in the legend to Figure 1 were grown to early exponential phase in minimal medium. Thiamine was added and cells were fixed at hourly intervals. DNA content was determined by flow cytometric analysis of Sytox Green-stained cells. In each flow cytometry profile, the number of hours after addition of thiamine is indicated in the top left corner and the septation index is shown in the top right corner. The positions of 1C and 2C control peaks are indicated on the abscissas.

The DE286AA mutant displayed an S-phase arrest phenotype (Figure 2). After the addition of thiamine, the septation index decreased by >80% within one cell cycle and remained low throughout the experiment, indicating that cell cycle progression had ceased and, thus, that the checkpoint is intact. Consistent with this interpretation, DAPI staining of the cells at 7 hr after the addition of thiamine revealed a single intact nucleus and an elongated cellular morphology due to continued growth in the absence of division (Figure 3B). In addition, the majority of the DE286AA mutant cells maintained an ~2C DNA content throughout the course of the experiment, while the remaining cells accumulated with a DNA content between 1C and 2C, suggesting that the initiation function of cdc18⁺ is at least partially retained. Similarly, a 2C arrest phenotype with retention of checkpoint control has been observed previously in association with the temperature-sensitive cdc18-K46 allele and may represent a partial loss of DNA replication function (NASMYTH and NURSE 1981 ; KELLY et al. 1993 ).

View larger version (90K): In this window In a new window Download PPT slide   Figure 3. Nuclear morphology of cdc18 mutants. Cells fixed 7 hr after the addition of thiamine in the experiment shown in Figure 2 were stained with DAPI and observed using a fluorescence microscope. (a) cdc18+ control. Wild-type cells form a single septum between two well-separated nuclei. (b) cdc18-DE286AA. Cells contain a single intact nucleus and exhibit an elongated morphology due to continued growth in the absence of division. (c) cdc18-K205A. The arrow indicates a "cut" cell resulting from formation of a septum across a single unreplicated nucleus. (d) cdc18-K205A. The arrow indicates a cell that has segregated its unreplicated DNA intact, resulting in the production of an anucleate cell. The bottom right cell has segregated its DNA asymmetrically, resulting in the production of two daughter cells with <1C DNA content.

The K205A mutant strain exhibited a mixed phenotype (Figure 2). By 4 hr after the addition of thiamine, the formation of a 1C peak was observed. At later times, cells with a <1C DNA content accumulated. However, even at 7 hr after the addition of thiamine, a significant percentage of the population retained a 2C DNA content and an additional fraction of the population accumulated with DNA contents between 1C and 2C. Consistent with the flow cytometry profile, the septation index decreased throughout the course of the experiment, but did not reach zero. In addition, DAPI staining revealed that only 25% of the cells exhibited abnormal nuclear morphology at the 7-hr timepoint (Figure 3C and Figure D). Thus, the mixed phenotype appears to be a composite of the null and S-phase arrest phenotypes. In addition, the difference in terminal phenotypes exhibited by the DE286AA and K205A mutants suggests that nucleotide binding and hydrolysis are important for distinct aspects of Cdc18 function.

The phenotypes exhibited by the nucleotide-binding motif and cdc18 mutants are cell-cycle-dependent:
The mixed phenotype exhibited by the nucleotide-binding motif mutant appears to be a composite of the null and S-phase arrest phenotypes; some cells in the population exhibit a null phenotype and the remainder are arrested with a 1C–2C DNA content. One possible explanation for this phenomenon is that the phenotype exhibited by an individual cell may be dependent on cell-cycle position at the time that wild-type cdc18⁺ expression is repressed. This model predicts that, in a synchronized culture, the K205A mutant will exhibit a homogeneous terminal phenotype. To investigate the nature of the mixed phenotype, mutant and control strains were synchronized in S phase by treatment with hydroxyurea (HU), a DNA synthesis inhibitor. After 3 hr, thiamine was added to the cultures to repress expression of the plasmid-borne copy of wild-type cdc18⁺. Thirty minutes later the HU was removed by washing in minimal medium containing thiamine and washed cells were inoculated into fresh thiamine-containing medium. Samples of each culture were removed and fixed before the addition of HU, after 3 hr in the presence of HU, and at intervals after release from the HU arrest. Fixed cells were examined by flow cytometry to determine DNA content and by light microscopy to determine the septation index.

As expected, the asynchronous wild-type control culture had a 2C DNA content before the addition of HU (Figure 4). After 3 hr in the presence of HU, the DNA content was ~1C and the septation index decreased by ~90%, indicating that the HU arrest was effective. By 1 hr after the removal of hydroxyurea, most of the cells had recovered from the arrest and progressed through S phase, as indicated by the observed 2C DNA content. At 2 hr after the removal of HU, the septation index increased as the cells completed mitosis and entered the subsequent cell cycle. Throughout the remainder of the experiment, the cells maintained a 2C DNA content and the septation index fluctuated slightly as the cells progressed through subsequent cycles in a partially synchronous fashion.

View larger version (26K): In this window In a new window Download PPT slide   Figure 4. Terminal phenotypes of cdc18 mutants in a culture synchronized by treatment with HU. The haploid strains described in the legend to Figure 1 were grown to early exponential phase in minimal medium and were then incubated in the presence of HU for 3 hr. Thiamine was added, and after 30 min HU was removed by washing in medium containing thiamine. Cells were fixed before the addition of HU (Exp, bottom), after 3 hr in the presence of hydroxyurea (+HU), and at hourly intervals after removal of HU (t = 1–7). DNA content was determined by flow cytometric analysis of Sytox Green-stained cells. In each panel, the time of fixation is indicated in the top left corner and the septation index is shown in the top right corner.

The empty vector control integrant also displayed a 1C DNA content and a decreased septation index in the presence of HU (Figure 4). By 1 hr after the removal of HU, most of the cells had synthesized a 2C DNA complement, confirming that continued expression of cdc18⁺ is not required for completion of S phase after release from an HU block (MUZI-FALCONI et al. 1996A ). At 2 hr after removal of HU, the septation index increased significantly as the cells completed mitosis. The accumulation of cells with a 1C DNA content was also observed at this time as the cells progressed into the next cell cycle and arrested at the beginning of S phase due to an inability to initiate DNA replication. At later times, cells with <1C DNA content are observed. Thus, consistent with the homogeneous null phenotype exhibited in an asynchronous culture, the empty vector control displays the null phenotype when expression of wild-type cdc18⁺ is repressed at the HU arrest point.

The nucleotide-hydrolysis motif mutant displayed the S-phase arrest phenotype under these conditions. After 3 hr in the presence of HU, the majority of the DE286AA cells displayed a 1C DNA content (Figure 4). By 1 hr after the removal of HU, most of the cells displayed a 2C DNA content. After 2 hr, the septation index increased as the cells completed mitosis and entered the subsequent cell cycle, and the accumulation of cells with a 1C–2C DNA content was observed. The flow cytometry profile did not significantly change throughout the remainder of the experiment, although the septation index decreased and elongated cells were visible at later times. Thus, consistent with the S-phase arrest phenotype exhibited in an asynchronous culture, the nucleotide-hydrolysis motif mutant displays a 1C–2C arrest phenotype when expression of wild-type cdc18⁺ is repressed at the HU arrest point.

Finally, the phenotype exhibited by the nucleotide-binding motif mutant was determined. The HU arrest of the K205A mutant culture was judged to be effective based on DNA content and septation index (Figure 4). By 1 hr after release from the HU block, the majority of the cells displayed a 2C DNA content. At 2 hr after removal of HU, the septation index increased as the cells completed mitosis and entered the subsequent cell cycle. The accumulation of cells with a 1C DNA content was also observed at this time as the cells arrested at initiation of S phase in the subsequent cycle. At later times, cells with <1C DNA content are observed. Thus, in contrast to the mixed phenotype exhibited in an asynchronous culture, the nucleotide-binding motif mutant displays a homogeneous null phenotype when expression of wild-type cdc18⁺ is repressed at the HU-arrest point and the terminal phenotype exhibited by this mutant is cell cycle dependent.

As an alternative method for synchronizing cultures, we constructed cdc18 mutant and control strains carrying the temperature-sensitive cdc25-22 allele, which confers a G₂/M arrest upon shift to the restrictive temperature. Cultures were grown in minimal medium at the permissive temperature and were arrested by incubation at the restrictive temperature for 4 hr. Thiamine was then added to the cultures to suppress expression of the plasmid-borne copy of wild-type cdc18⁺ and the cultures were shifted to the permissive temperature. Samples of the cultures were removed and fixed at 15-min intervals for 270 min after return to the permissive temperature. Fixed cells were examined by flow cytometry to determine DNA content and by light microscopy to determine the septation index.

As expected, all of the asynchronous cultures exhibited a 2C DNA content (Figure 5). After 4 hr at 36°, the septation indices decreased to <1% (data not shown) and the cells retained a 2C DNA content. Note that the position of the 2C peak is shifted to the right due to elongation of the cells and continued synthesis of mitochondrial DNA during the cell cycle arrest (SAZER and SHERWOOD 1990 ). After return to the permissive temperature, the wild-type cdc18⁺ control strain progressed synchronously through the first mitosis and septation peaked at 75–90 min (Figure 5). A second peak of septation was observed at 195 min as the cells progressed through the subsequent cell cycle in a partially synchronous fashion. After 270 min, the wild-type control cells had returned to a normal size and exhibited a 2C DNA content, consistent with the fact that they continued to cycle. In contrast, the nucleotide-hydrolysis motif mutant completed the first cell cycle and septated at 75–90 min after release from the cdc25^ts arrest, but did not exhibit a second peak of septation. Examination of cells fixed 270 min after shift to the permissive temperature revealed that this mutant arrested with an elongated cellular morphology (data not shown) and an ~2C DNA content (Figure 5).

View larger version (25K): In this window In a new window Download PPT slide   Figure 5. Terminal phenotypes of cdc18 mutants in a culture synchronized using a cdc25ts allele. The cdc25-22 allele was introduced into the haploid strains described in the legend to Figure 1. The resulting strains were grown to early exponential phase in minimal medium at the permissive temperature of 25° and were then incubated at the restrictive temperature of 36° for 4 hr. Thiamine was added and the cultures were returned to 25° for 270 min (Arrest). Cells were fixed before the cultures were shifted to 36° (Exp), after 4 hr at 36° (36°), and at 15-min intervals after return to 25°. The top section shows the flow cytometry profiles of Sytox Green-stained cells. The graph in the bottom shows the septation index of the cultures as a function of time after return to 25° in the presence of thiamine.

The nucleotide-binding motif mutant and empty vector control strains also underwent a cell-cycle arrest after expression of wild-type cdc18⁺ was repressed. The septation index peaked at 75–90 min after shift to the permissive temperature and remained low throughout the rest of the experiment. Examination of cells fixed 270 min after release from the cdc25^ts arrest revealed that these strains arrested with an elongated cellular morphology (data not shown) and the majority of cells exhibited an ~2C DNA content. Thus, the nucleotide-binding motif mutant and empty vector control strains display a nearly homogeneous arrest phenotype when expression of wild-type cdc18⁺ is repressed at the cdc25^ts-arrest point.

Mutants exhibiting the 1C–2C arrest phenotype are arrested in S phase:
The nucleotide-hydrolysis motif mutant exhibits a 1C–2C DNA content at its arrest point, indicating that the cells are arrested in S phase. The nucleotide-binding motif mutant and empty vector control strains arrest with a nearly homogeneous phenotype and an ~2C DNA content when expression of wild-type cdc18⁺ is repressed at the cdc25^ts-arrest point. This observation is consistent with an arrest in late S or G₂ phases of the cell cycle. To confirm that the DE286AA mutant arrests in S phase and to determine whether DNA replication is complete at the 2C arrest point, chromosomal DNAs from the DE286AA and cdc25^ts, K205A, or empty vector strains were analyzed by pulsed-field gel electrophoresis (Figure 6). When subjected to pulsed-field gel electrophoresis, chromosomal DNAs isolated from certain mutants that are defective for DNA replication are unable to enter the gel (HENNESSY et al. 1991 ; WASEEM et al. 1992 ). It has been proposed that the presence of residual replication intermediates in these mutants results in topological constraints on the DNA, thus preventing it from leaving the well.

View larger version (61K): In this window In a new window Download PPT slide   Figure 6. Pulsed-field gel electrophoresis of chromosomal DNA prepared from cdc18 mutant strains. (a) A haploid strain constructed as described in the legend to Figure 1 and carrying the DE286AA mutant cdc18 allele was grown to early exponential phase in minimal medium. Thiamine was added and incubation was continued for 5.5 hr. Exponentially growing and HU-arrested cultures of the wild-type cdc18+ control integrant were also prepared. Chromosomal DNAs were isolated in agarose plugs and were subjected to pulsed-field electrophoresis. P indicates the position of the agarose plugs at the origin of electrophoresis. I, II, and III indicate the positions of the S. pombe chromosomes 1, 2, and 3, respectively. (b) Haploid strains constructed as described in the legend to Figure 5 and carrying the K205A mutant cdc18 allele, wild-type cdc18+, or an empty vector construct were grown to early exponential phase in minimal medium at 25°. The cultures were incubated at 36° for 4 hr to arrest them at G2/M. Thiamine was then added and the cultures were returned to 25° for 270 min. Chromosomal DNAs were isolated in agarose plugs and were subjected to pulsed-field electrophoresis.

Chromosomal DNA was prepared from the DE286AA mutant after 5.5 hr in the presence of thiamine. The cells showed an 80% decrease in septation index and a slightly elongated cellular morphology at the time of harvest, indicating that the majority of cells in the population had undergone a cell-cycle arrest. A 1C–2C DNA content was confirmed by flow cytometric analysis (data not shown). Consistent with the 1C–2C DNA content, chromosomal DNA isolated from the DE286AA strain did not enter the gel (Figure 6A). In control experiments, chromosomal DNA isolated from an exponentially growing wild-type culture was resolved on the gel, while chromosomal DNA isolated from a wild-type culture arrested in S phase by treatment with HU did not enter the gel. These observations confirm that the nucleotide-hydrolysis motif mutant arrests in S phase.

Similarly, chromosomal DNAs were prepared from the K205A and empty vector strains in the cdc25^ts background. Cultures were arrested at G₂/M by incubation at the restrictive temperature for 4 hr. Thiamine was added to repress expression of the plasmid-borne copy of wild-type cdc18⁺ and the cultures were shifted to the permissive temperature for 270 min. At the time of harvest, both strains had an elongated cellular morphology and a 2C DNA content (data not shown). As in the case of the DE286AA mutant, the chromosomal DNAs did not enter the gel (Figure 6B). In control experiments, chromosomal DNA isolated from the wild-type cdc18⁺ integrant in the cdc25^ts strain background was resolved on the gel, confirming that incubation at the restrictive temperature is not sufficient to prevent completion of DNA replication after release to the permissive temperature. These observations indicate that the K205A mutant and the empty vector control strains are in S phase when they are arrested with an ~2C DNA content.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this study, we have shown that mutants containing disruptions in the conserved Walker A and B box motifs of cdc18⁺ are inviable, suggesting that the biochemical activities that are commonly associated with these motifs are required for the essential function of Cdc18. The Walker A box is required for nucleotide binding by many proteins (ROZEN et al. 1989 ; HABER and WALKER 1991 ; PAUSE and SONENBERG 1992 ; KLEMM et al. 1997 ). Structural studies have indicated that the A box sequence, GXXGXGKT, forms a phosphate-binding loop in which the positively charged lysine residue interacts directly with the negatively charged phosphate groups of the bound nucleotide (FRY et al. 1986 ; PAI et al. 1990 ; STORY and STEITZ 1992 ). Mutation of the conserved lysine to threonine in the A box motif found in S. cerevisiae Orc1 eliminated ATP binding detected by both gel-filtration assay and 8-azido-ATP crosslinking (KLEMM et al. 1997 ). This motif is conserved between Orc1 and Cdc18 and lies in a region of Orc1 that shares ~50% similarity with amino acids 169 to 435 of Cdc18 (BELL et al. 1995 ). Additionally, mutation of lysine to alanine in the Salmonella typhimurium MutS mismatch repair protein increased the K_m for ATP by ~35-fold over the wild-type protein (HABER and WALKER 1991 ). This is the same amino acid change introduced in the cdc18 K205A allele. Taken together, these studies suggest that the phenotype of the K205A mutant is due to a defect in nucleotide binding; however, we have as yet been unable to detect binding of purine nucleotide triphosphates to purified recombinant Cdc18 in vitro, possibly because other proteins are required to activate nucleotide binding (C. L. SMITH and G. S. MARTIN, unpublished results).

It has been proposed that the Walker B box, DEXX, is required specifically for nucleotide hydrolysis. Crystal structure data indicate that the negatively charged aspartate residue of this motif interacts with the Mg²⁺ ion required for nucleotide hydrolysis (FRY et al. 1986 ; PAI et al. 1990 ; STORY and STEITZ 1992 ). Mutational analyses also support this hypothesis. Mutation of the DEAD sequence of mammalian translation initiation factor eIF4A to NEAD or DQAD had no effect on nucleotide binding as measured by UV crosslinking assay, but eliminated detectable ATPase activity (PAUSE and SONENBERG 1992 ). Similarly, biochemical characterization of the DNA-dependent ATPase activity of a DEXX to NQXX mutant of the Escherichia coli DNA helicase II protein demonstrated a k_cat that was <0.5% of that of the wild-type protein, while the apparent K_m was not affected (BROSH and MATSON 1995 ). Finally, changing the equivalent DE of the S. cerevisiae Upf1 protein to AA also abrogates ATPase activity. Wild-type Ubf1p:RNA complexes are dissociated as a consequence of ATP binding by Ubf1p. The DE to AA mutant retains this property while a Walker A box mutant (GKT to GQT) does not, suggesting that the DE to AA mutant is functional for nucleotide binding (WENG et al. 1996 ). This is the same mutation introduced in the cdc18 DE286AA allele, suggesting that nucleotide hydrolysis is also required for Cdc18 function.

It is particularly interesting that the K205A and DE286AA mutants have different terminal phenotypes. In HU-synchronized cultures, the K205A (nucleotide-binding site) mutant did not enter S phase in the absence of wild-type cdc18⁺ expression. In contrast, the DE286AA (nucleotide-hydrolysis) mutant arrested in S phase with incompletely replicated DNA and an elongated cellular morphology. It is unlikely that the DNA synthesis that occurs in this mutant is due solely to the activity of residual wild-type Cdc18, because DNA replication occurs in synchronized cultures and under conditions where an empty vector control strain arrests before S phase. In addition, it is unlikely that residual wild-type Cdc18 activity contributes to the phenotype observed when wild-type cdc18⁺ expression is repressed at the HU arrest point, because the Cdc18 protein is normally present during G₁ and is destabilized upon phosphorylation by Cdc2 during other phases of the cell cycle (MUZI-FALCONI et al. 1996A ; JALLEPALLI et al. 1997 ). However, it is possible that both the mutant and wild-type proteins are necessary for the DNA replication observed in the DE286AA mutant.

A phenotype similar to that of the DE286AA mutant has been previously observed for the temperature-sensitive cdc18-K46 mutant and has been proposed to result from a partial loss of a single Cdc18 function that is required for DNA replication (NASMYTH and NURSE 1981 ; KELLY et al. 1993 ). A partially functional mutant would be expected to initiate DNA replication at some origins, but the reduced number of origins that fire would be insufficient to completely replicate the genome. By this "single-function" model, the observed difference in phenotypes between the K205A and DE286AA mutants could be due to quantitative differences in the amount of residual nucleotide-binding and/or hydrolytic activity of the mutant proteins. The K205A mutation is predicted to have a more dramatic effect on nucleotide binding and/or hydrolysis than the DE286AA mutation and therefore may cause more severe defects in initiation of DNA replication, leading to a difference in phenotypes.

An alternative possibility is that Cdc18 has two distinct functions in the cell cycle, or that it performs its function in two steps that can be temporally separated. In this model, the S-phase arrest phenotype would be displayed by mutants that retain the first function of Cdc18, but are defective for a second, undefined function. The difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis mutants is consistent with what is known of nucleotide-hydrolyzing "switch" proteins. These proteins alternate between two states: a constrained state with bound nucleotide triphosphate, and a relaxed state with bound nucleotide diphosphate, the two forms having different affinities for effectors and regulators (reviewed in BOGUSKI and MCCORMICK 1993 ). By analogy, the nucleotide triphosphate-bound form of Cdc18 could be required for the first function of Cdc18, while the nucleotide diphosphate-bound form is required for the second function. This is similar to the model recently proposed for Orc1. ATP binding by Orc1 is required for the ORC to bind DNA. Once bound to DNA, ATP remains stably associated with Orc1 and is not hydrolyzed, although Orc1 ATPase activity is detectable in vitro. These observations led to the proposal that nucleotide binding by Orc1 regulates the association of ORC with DNA, while nucleotide hydrolysis is required for some other aspect of ORC function, such as origin firing or disassembly of the prereplication complex (KLEMM et al. 1997 ). The genetic data presented here and the sequence similarity between Orc1 and Cdc18 suggest that these two proteins function by a common mechanism.

The nucleotide-binding mutant exhibits a mixed phenotype when expression of wild-type cdc18⁺ is repressed in an asynchronous culture and arrests with a uniform phenotype when cdc18⁺ is repressed in synchronized cultures. There are two possible explanations for the cell-cycle dependence of the K205A mutant phenotype. The first is that the two postulated functions of Cdc18 are executed at different times. In this case, the phenotype exhibited by an individual cell that is defective for both Cdc18 functions would be dependent on which function is required first after depletion of wild-type Cdc18. Thus, cells that have not executed the first or second Cdc18 functions at the time of wild-type Cdc18 depletion would arrest at a termination point that reflects failure to complete the first Cdc18 function (the null phenotype). Conversely, cells that have executed the first Cdc18 function (nucleotide binding) but not the second (nucleotide hydrolysis) at the time of wild-type Cdc18 depletion would arrest at a termination point that reflects failure to complete the second function (an S-phase arrest). The nucleotide-binding mutant displays a homogeneous null phenotype when wild-type cdc18⁺ expression is repressed at the HU arrest point, suggesting that if Cdc18 does execute two temporally separable functions, both have been executed before this point in the cell cycle. Like the nucleotide-binding mutant, the empty vector control strain exhibited a nearly homogeneous S-phase arrest phenotype when expression of wild-type cdc18⁺ was repressed at the cdc25^ts-arrest point. It is possible that, under these conditions, the first cdc18⁺ function is completed by the plasmid-borne copy of cdc18⁺ at or before the arrest; however, it is unlikely that this reflects the situation in the wild-type cell, because both the cdc18⁺ transcript and protein are known to be cell-cycle regulated and are only detected at G₁/S during a normal cell cycle (KELLY et al. 1993 ; MUZI-FALCONI et al. 1996A ). Consistent with the possibility that constitutive expression of wild-type cdc18⁺ and/or inactivation of cdc25-22 could allow for execution of the first function at an aberrant point in the cell cycle, recent evidence has indicated a role for the Cdc2 kinase in the destabilization of Cdc18 (JALLEPALLI et al. 1997 ; LOPEZ-GIRONA et al. 1998 ). Thus, inactivation of Cdc25 could lead to aberrant stabilization of Cdc18 at G₂/M.

A second possible explanation for the cell-cycle dependence of the K205A mutant phenotype is that, following repression of cdc18⁺ expression, a small fraction of wild-type Cdc18 could persist until the point in the cell cycle at which cdc18⁺ function is executed. Thus, the extent to which Cdc18 is depleted would be affected by cell cycle position at the time when cdc18⁺ expression is repressed and by the genetic background of the strain, which can affect the stability of Cdc18 (JALLEPALLI et al. 1997 ; LOPEZ-GIRONA et al. 1998 ). It is unlikely that the persistence of wild-type Cdc18 by itself could account for the cell-cycle dependent phenotype of the nucleotide-binding motif mutant, because, in the wild-type background, asynchronous cultures of the null mutant never displayed a mixed phenotype. However, it is possible that residual wild-type Cdc18 could cooperate with the mutant protein, perhaps in some multimeric complex, resulting in the DNA replication observed in the K205A mutant strain.

In summary, the data presented here indicate that both nucleotide binding and hydrolysis are required for Cdc18 function and are consistent with a two-step model in which nucleotide binding and nucleotide hydrolysis are required for execution of distinct functions. While this article was in preparation, PERKINS and DIFFLEY 1998 reported the properties of similar Walker A and B Box mutants of Cdc6, the S. cerevisiae homolog of fission yeast Cdc18. Footprinting and chromatin precipitation studies suggested that the A box mutation affected Cdc6 binding to origins of replication, while the B box mutation affected Mcm recruitment. If these conclusions are applicable to Cdc18, they would provide strong support for the two-function model.

	ACKNOWLEDGMENTS

We are very grateful to Susan Forsburg for advice on flow cytometry, to Mike Botchan for many insightful discussions, and to Jasper Rine, Bill Jackson, Franz Hofer, Andrew Dillin, and Yaron Hakaka for critical reading of this manuscript. We also thank Kathy Gould for providing strains. This research was supported by grant CB107 from the American Cancer Society and grant CA17452 from the National Institutes of Health. D. DeRyckere was supported by a predoctoral fellowship from the Howard Hughes Medical Institute.

Manuscript received September 8, 1998; Accepted for publication January 15, 1999.

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