CDC45 Is Required in Conjunction with CDC7/DBF4 to Trigger the Initiation

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12521–12526, November 1997 Genetics

CDC45 is required in conjunction with CDC7͞DBF4 to trigger the initiation of DNA replication

JULIA C. OWENS,CORRELLA S. DETWEILER, AND JOACHIM J. LI*

Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414

Communicated by Ira Herskowitz, University of California, San Francisco, CA, September 16, 1997 (received for review July 25, 1997)

ABSTRACT The initiation of DNA replication in Saccha- S phase (23). Formation of the pre-RC, although necessary, is romyces cerevisiae requires the protein product of the CDC45 not sufficient to initiate replication in yeast. Cells must also gene. We report that although Cdc45p is present at essentially pass through the G1 commitment point START before they constant levels throughout the cell cycle, it completes its can execute a second step that presumably activates the initiation function in late G1, after START and prior to DNA pre-RC and triggers the initiation of DNA replication (re- synthesis. Shortly after mitosis, cells prepare for initiation by viewed in refs. 24–26). This step is thought to require two assembling prereplicative complexes at their replication ori- kinases, Cdc7p (12) and Cdc28p (15), in association with their gins. These complexes are then triggered at the onset of S respective regulatory subunits, Dbf4p (12) and Clb5p͞Clb6p phase to commence DNA replication. Cells defective for (27, 28). The entry into S phase correlates with replacement of CDC45 are incapable of activating the complexes to initiate the pre-RC by an ORC-like postreplicative complex (post-RC) DNA replication. In addition, Cdc45p and Cdc7p͞Dbf4p, a (16). This transition presumably reflects the simultaneous kinase implicated in the G1͞S phase transition, are dependent activation and disassembly of the pre-RC during initiation and on one another for function. These data indicate that CDC45 we refer to this transition as the triggering of the pre-RC. functions in late G1 phase in concert with CDC7͞DBF4 to CDC45 is an essential gene required for DNA replication trigger initiation at replication origins after the assembly of (29–31). Cdc45p has been localized to the nucleus (29) and the prereplicative complexes. CDC45 mRNA expression is periodic, peaking during the G1͞S transition (32). Three different experiments have suggested it Maintenance of genome integrity is essential for cell viability. functions in the initiation of replication. First, mutations in Two events in the cell cycle critical to this task are the CDC45 have been shown to interact genetically with mutations replication of chromosomes during S phase and their segre- in several initiation components, including members of the gation in mitosis. During S phase, the cell must assure not only MCM family and components of ORC (31–33). Second, that replication is completed before mitosis, but also that each mutant cdc45 cells lose plasmids at an elevated frequency in a origin initiates replication no more than once per cell cycle. manner that can be suppressed by the addition of multiple Saccharomyces cerevisiae has proven to be an exceptionally origins to the plasmids (29, 31, 32). Finally, analysis of repli- tractable system in which to dissect the mechanism and cation intermediates in cdc45-1 cells shows a reduced fre- regulation of replication initiation. Powerful genetic tech- quency of firing at individual origins of replication (31). niques in addition to well-defined origins of replication have We have studied CDC45 further to understand its role in facilitated the identification of many yeast components re- replication initiation. We show that despite the periodicity of quired for the initiation of DNA replication. These include the its mRNA expression, Cdc45p is present at relatively constant origin recognition complex (ORC) that binds to yeast origins levels throughout the cell cycle, and that it completes its (1–6), a family of structurally and functionally similar proteins initiation function in late G1, after START and prior to called MCM (minichromosome maintenance) proteins (7–10), elongation of DNA replication. Cells defective for CDC45 Cdc6p (11), and two kinases, Cdc7p (12–14) and Cdc28p (15). function are unable to trigger pre-RCs once they are formed. Genomic footprint analysis of the proteins assembled at In addition, Cdc45p and Cdc7p kinases are dependent on each yeast origins during the cell cycle has suggested a two-step other for execution of their replication functions. A similar model for the initiation of DNA replication (16). Many of the interdependent relationship was observed between Cdc45p initiation proteins mentioned above have been implicated in and Dbf4p, the regulatory subunit for Cdc7p kinase. These one or both of these steps. ORC is believed to be bound to data lead to the conclusion that CDC45 functions in conjunc- origins throughout the cell cycle. In the first step, which occurs tion with Cdc7p͞Dbf4p to trigger pre-RCs during the initiation shortly after mitosis, additional proteins are thought to join of DNA replication. ORC to form prereplicative complexes (pre-RCs) at origins in preparation for S phase (16). One of these proteins might be MATERIALS AND METHODS Cdc6p because it is required for both assembly and maintenance of the pre-RC (17, 18). Other candidates include the six Media and Budding Index. Yeast extract͞peptone (YEP) members of the MCM family of proteins (MCM2, MCM3, medium (34) was supplemented with 2% dextrose (YEPD). MCM5͞CDC46, CDC47, CDC54, and MCM6) (19–21). In Unless otherwise stated, cells were grown at 30°C. To arrest yeast, several of these proteins have been shown to shuttle cells, ␣-factor was used at 50 ng͞ml (for bar1 cells) or 10 ␮g͞ml between the cytoplasm and nucleus, entering the nucleus (for BAR1 cells); hydroxyurea (HU) was used at 0.2 M. For coincident with pre-RC formation (7, 8, 16, 22). In Xenopus analysis of budding index, cells with bud diameter less than replication extracts, MCM homologs appear to be loaded onto 50% that of the mother cell were scored as small-budded cells, chromatin in an XCdc6p-dependent manner in preparation for whereas those with a bud diameter greater than 50% that of

The publication costs of this article were defrayed in part by page charge Abbreviations: ORC, origin recognition complex; MCM, minichromosome maintenance; pre-RC, prereplicative complex; post-RC, post payment. This article must therefore be hereby marked ‘‘advertisement’’ in replicative complex; HU, hydroxyurea; HA, hemagglutinin. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed at: Department of © 1997 by The National Academy of Sciences 0027-8424͞97͞9412521-6$2.00͞0 Microbiology, Box 0414, University of California, San Francisco, CA PNAS is available online at http:͞͞www.pnas.org. 94143-0414. e-mail: [email protected].

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the mother cell were scored as large-budded cells. In our RESULTS strains the appearance of small buds coincides with the onset of replication (18). Cdc45p Is Maintained at Constant Levels in Cycling Cells. Plasmids and Strains. The cdc45-1 yeast strain used to order Previous studies have shown that CDC45 mRNA expression is CDC45 function relative to START and replication elongation periodic, with maximal levels appearing at the G1͞S phase is YJL556 (DBY2027, MATa cdc45-1 ade2-1 lys2-801 leu2-3, boundary (32). To determine whether Cdc45p levels fluctu- 112 ura3-52). The related wild type strains used for controls ated in parallel with mRNA levels, we constructed a strain in were YJL1086 (DBY1705, MAT␣ leu2-3, 112 ura3-52 lys2-801) which three tandem copies of the HA epitope (36) were fused and YJL1085 (DBY640, MATa ade2-1 gal- mal-). The strain to the C terminus of the endogenous protein (Cdc45p-HA3). used to functionally order CDC45 and CDC7 is YJL1907 The tagged protein appears to function normally because this (MATa cdc45-1 cdc7-4 leu2-3, 112 ura3-52 ade2-1 lys2-801 strain grew equivalently to a wild-type strain and progressed trp1-289). The strain used to functionally order CDC45 and normally through the cell cycle as indicated by its budding DBF4 is YJL1908 (MATa cdc45-1, dbf4-1, leu2-3, 112, trp1-289, index and flow cytometry profile (data not shown). CDC45- ura3-52, bar::LEU2). The strains used for genomic footprinting HA3 cells were synchronously released from an ␣-factor- are YJL1496 (MATa cdc45-1 ade2-1 lys2-801 leu2-3, 112 induced G1 arrest, and the amount of the tagged protein was ura3-52 bar1::LEU2) and YJL310 (MATa leu2-3, 112 ura3-52 assessed by immunoblotting with anti-HA mAbs. Good syn- trp1-289 bar1::LEU2). Epitope-tagged CDC45 was produced chrony was maintained for two cell cycles as monitored by by first cloning a blunt-ended BsaI–SphI CDC45 genomic budding index (Fig. 1A) and flow cytometry (data not shown). fragment into the XhoI and NotI sites of pRS306. The sequence With the exception of the earliest time points, the level of 5Ј-GGCGGCCGCGCACCGGTG-3Ј containing the NotI and Cdc45p-HA3 was relatively constant during both cell cycles SgrAI restriction sites was inserted by oligo-directed mutagen- (varying less than 2.5-fold after normalization for differences esis (35) immediately 5Ј of the CDC45 stop codon and the in lane loading). The elevated protein levels present in G1 mutagenesis confirmed by sequencing. Into the NotI site, a phase of the second cell cycle (100 and 110 min) suggested that NotI fragment encoding three tandem copies of the hemag- the low amounts observed at the beginning of the time course (0 and 10 min) did not represent normal G1 phase levels but glutinin epitope (HA)3 (36) was inserted in-frame with the CDC45 ORF, yielding the plasmid pJO05. The tagged CDC45 arose from the prolonged ␣-factor arrest used to synchronize gene was substituted for the wild-type copy by two-step gene the cells. replacement (34) yielding yeast strain YJL1906 (MATa To confirm this notion, cells were synchronously released from an S phase arrest induced by HU and allowed to progress CDC45-HA3 ura3-52 trp1-289 leu2-3, 112 bar1::LEU2). The desired replacement was confirmed by Southern blot analysis into an ␣-factor block in the next cell cycle (Fig. 1B). Within (35). 120 min of release from the HU block, cells were fully arrested Yeast Protein Preparations for Western Blot Analysis. Cells in G1 as monitored by flow cytometry (data not shown), but still maintained the elevated levels of Cdc45p seen in cycling (4 ml) at OD600 0.5–1.0 were pelleted and lysed by vortex mixing and boiling with 200 ␮l 0.5-mm glass beads (Biospec cells. Eventually, after more prolonged arrest, protein levels Products, Bartlesville, OK) and 150 ␮l SDS͞PAGE loading buffer (5% glycerol͞0.5% SDS͞64 mM Tris⅐Cl͞120 mM DTT) with protease inhibitors (4 mM EDTA͞1 mM phenylmethyl- sulfonyl fluoride͞1 ␮g/ml leupeptin͞10 ␮g/ml aprotinin͞2mM benzamidine͞1 ␮g/ml pepstatin A). The soluble protein was quantified by Bradford assay (Bio-Rad) by using BSA fraction V (Sigma) as a standard. Forty micrograms of each sample were separated on SDS͞PAGE and transferred to a Micron Separations (Westboro, MA) Nitrobind 0.22-␮m membrane. The membrane was probed with 12CA5 anti-HA mAb diluted at 1:2,000 [Babco ascites (Babco, Richmond, CA)] or anti- Sec61 antibodies diluted at 1:6,000 (antiserum a gift of Sylvia Sanders, Massachusetts Institute of Technology and used as a loading control), followed by horseradish peroxidase- conjugated goat anti-mouse or donkey anti-rabbit secondary antibodies (Bio-Rad), respectively. Immunoblots were devel- oped with the Amersham ECL system. Functional Ordering and Flow Cytometry. Cells were released from arrests by filtering, washing with at least two volumes of medium and resuspending in fresh medium pre- warmed or precooled to the appropriate temperature and containing the appropriate drug. cdc45-1 arrests were obtained by incubation at 11°C for 16 h; cdc7-4 and dbf4-1 arrests were achieved by incubation at 38°C for 2.5–3 h. Arrests at START and prior to replication elongation were obtained by incubation of cells in the presence of ␣-factor and HU, respectively, FIG. 1. Cdc45p is present at constant levels in cycling cells. (A) for2hat30°C. Samples were taken for flow cytometry as CDC45-HA3 cells were synchronously released from an ␣-factor arrest. described (18). Whole-cell extracts were made at various time points and total cell Genomic Footprinting. Genomic footprinting of the 2- protein extracts were made, resolved by SDS͞PAGE, and immunob- micron origin of replication was performed essentially as lotted with antibodies against the HA epitope (Lower). Blots were described (37, 38). The exposure time used for the cdc45-1 reprobed with anti-Sec61p antibodies as a loading control (18). Cell synchrony was evaluated by determining percent of unbudded, small- genomic footprints was five times that of the CDC45 footprints. budded, and large-budded cells. (B) CDC45-HA3 cells were synchro- This adjustment was necessary because cdc45-1 cells, which are nously released from a HU arrest into medium containing ␣-factor. defective in maintaining plasmids (29, 31, 32), presumably Total cell protein extracts were analyzed as described above. Cells contain, on average, a lower level of 2-micron plasmids than reached the ␣-factor block 120 min after release from HU as moni- CDC45 cells. tored by flow cytometry (data not shown). Downloaded by guest on September 30, 2021 Genetics: Owens et al. Proc. Natl. Acad. Sci. USA 94 (1997) 12523

dropped to the low levels seen at 0 min in Fig. 1A. Thus, we However, we cannot rule out a role for CDC45 in the efficient conclude that Cdc45p levels remain constant in cycling cells. and complete progression of replication forks during elonga- Because the protein levels are maintained despite periodic tion. fluctuations in CDC45 mRNA levels(32), we also infer that Cdc45p Is Required After START for DNA Replication. Cdc45p is relatively stable in these cells. Previous experiments have suggested that CDC45 function is Cdc45p Completes its Replication Function Before DNA needed after START for optimal DNA replication (31). To Elongation Occurs. To examine when Cdc45p is needed for determine whether CDC45 function is actually essential for DNA replication, we determined the order in which CDC45 replication after START, we released cdc45-1 and CDC45 cells functions relative to two cell cycle events: the elongation phase from a START arrest imposed by ␣ factor at 30°C into medium of DNA replication in S phase and the passage through at the cdc45-1 restrictive temperature of 11°C. As shown in Fig. START in G1 phase. Experiments with similar aims have been 3A, cdc45-1 cells maintained a 1C DNA content 12 h after described (31) but were complicated by incomplete inactiva- release from ␣ factor, whereas the CDC45 wild-type strain tion of CDC45 function and inability to recover cycling cells replicated and acquired a 2C DNA content within 6 h following from the cdc45-1 arrest (see Discussion). release. The cdc45-1 cells did release from the ␣-factor arrest, In our first set of experiments we asked whether CDC45 because Ͼ90% of the cells were observed as large-budded cells function is necessary for replication once cells have entered S (data not shown). This result indicates that Cdc45p function is phase. We released cdc45-1 and CDC45 cultures arrested in S required after START for replication and that execution of phase by HU, which blocks replication elongation, into the this function is dependent on passage through START. cdc45-1-restrictive temperature of 11°C and monitored DNA To determine whether passage through START depends on content by flow cytometry. As seen in Fig. 2A, both cdc45-1 completion of CDC45 function, we performed the reciprocal and CDC45 cells replicated after release from the HU block. experiment. cdc45-1 cells were arrested at 11°C and released This finding suggests that Cdc45p is not required for DNA into ␣-factor-containing medium at 30°C. Fig. 3B shows that elongation and executes its function independently of this step. replication occurred within 60 min after release, suggesting To determine whether replication elongation depends on that the passage through START does not depend on execu- execution of Cdc45p function, we performed the reciprocal tion of CDC45 function. This finding is consistent with the experiment. cdc45-1 cells were arrested at 11°C and released observation that cdc45-arrested cells have budded, an event into HU-containing medium at 30°C. Flow cytometry showed that requires passage through START. Together with results that cdc45-1 cells were unable to replicate after release from from the previous set of reciprocal shift experiments, these the 11°C block, retaining a 1C DNA content (Fig. 2B). In data indicate that CDC45 performs an essential replication contrast, cdc45-1 cells released at 30°C in the absence of HU function between START and DNA elongation. replicated within 40 min, confirming that the cdc45-1 arrest Cdc45p Is Necessary for Triggering of the Pre-RC. Current was reversible (Fig. 2C). Thus, we conclude that DNA elon- models (reviewed in refs. 24–26) suggest that initiation can be gation is dependent on prior execution of a CDC45 function, broken down into at least two steps: the assembly of pre-RCs consistent with Cdc45p playing a role in the initiation of DNA at replication origins shortly after mitosis and the triggering of replication (29, 31, 32). initiation during the G1͞S phase transition (16). The experi- It should be noted, however, that the cdc45-1 cells released ments described above are consistent with CDC45 having a from HU into 11°C replicated slightly slower than the wild- role in triggering initiation. To further explore this role, we type strain and that many of these cells did not proceed examined the genomic footprint at the 2-micron origin of through mitosis (data not shown). These observations might be replication in cdc45-1 and CDC45 strains. In wild-type cells, explained by impaired initiation at late replication origins changes in this footprint during the cell cycle are thought to resulting in incomplete replication and checkpoint arrest. reflect the assembly and disassembly of the pre-RC. During S,

FIG. 2. DNA elongation is dependent on CDC45 function. (A) FIG. 3. CDC45 function is dependent on START and is essential cdc45-1 and CDC45 cultures were arrested in S phase with HU- for DNA replication. (A) cdc45-1 and CDC45 cultures were arrested containing media at 30°C. At 0 h, the cultures were released from the in G1 phase with ␣-factor-containing medium at 30°C. At 0 h, the S phase arrest and shifted to 11°C. Samples were monitored flow cultures were released from the START arrest and shifted to 11°C. cytometry and budding index. (B)Acdc45-1 culture was arrested by Samples were monitored by flow cytometry and budding index. (B)A incubation in medium at 11°C, the cdc45-1-restrictive temperature. At cdc45-1 culture was arrested by incubation in medium at 11°C, the 0 min, the culture was shifted to 30°C and HU was added. Samples cdc45-1-restrictive temperature. At 0 min, the culture was shifted to were monitored by flow cytometry and budding index. (C) Samples 30°C and ␣ factor was added. Samples were monitored by flow were taken as in B, except no HU was added. cytometry and budding index. Downloaded by guest on September 30, 2021 12524 Genetics: Owens et al. Proc. Natl. Acad. Sci. USA 94 (1997)

G2, and M phases, this footprint resembles that of purified Genomic footprinting at this block reveals the presence of the ORC bound to the 2-micron origin and is most distinctly hypersensitive site (Fig. 4 A, lanes 11°C), consistent with the characterized by the presence of a hypersensitive site. ORC is disassembly of the pre-RC during initiation. In contrast, thus thought to be bound to the origin during these phases of cdc45-1 cells were unable to replicate and arrested as large- the cell cycle as part of a post-RC. In G 1, loss of the budded cells (confirming release from the ␣ factor block) hypersensitive site is the most dramatic change observed in the containing a 1C DNA content (Fig. 4 B Lower). The genomic genomic footprint and is indicative of pre-RC assembly. Dis- footprint at this arrest was virtually identical to the one assembly of the pre-RC is marked by reappearance of the observed at the ␣ factor arrest (Fig. 4 A, lanes 11°C), indicating post-RC with its signature hypersensitive site and tightly that the pre-RC had not disassembled and was still present at correlates with entry into S phase. Because of this correlation, the origin. The persistence of the pre-RC at the cdc45-1 block the pre-RC to post-RC transition is thought to signal the suggests that Cdc45p is needed to trigger initiation at repli- triggering of initiation at origins (16) and was used by us to cation origins. monitor the execution of the second initiation step. Cdc45p Functions in the Same Step as Cdc7p ͞Dbf4p. CDC45 and cdc45-1 cells were arrested with ␣ factor at the Mutations in CDC7 cause cells to arrest in late G 1 phase at the permissive temperature of 30°C. As expected, both strains last genetically defined point before S phase (11), suggesting arrested as unbudded cells with a 1C DNA content (Fig. 4 B that the kinase is required to trigger initiation. To determine Upper). Moreover, in both strains the absence of the ORC- the relative dependencies of CDC7 and CDC45, we performed induced hypersensitive site in the genomic footprint of the reciprocal shift experiments with a cdc45-1 cdc7-4 double 2-micron origin indicated the presence of the pre-RC (Fig. 4 A, mutant strain. This experiment takes advantage of the fact that lanes ␣f). Cells were then released from the ␣-factor arrest at cdc45-1 is a cold-sensitive allele and cdc7-4 is a temperature- 11°C in the presence of nocodazole for 9 h. During this time sensitive allele, allowing us to inactivate each gene indepen- CDC45 cells initiated and completed S phase, arresting at the dently of the other. nocodazole block with a 2C DNA content (Fig. 4 B Lower). To determine whether CDC45 function is needed after a CDC7 block, we released cdc45-1 cdc7-4 double mutant cells from a cdc7-4 arrest at 38°C into medium at either 11°C, the restrictive temperature for cdc45-1, or 27°C, the permissive temperature for both mutants. The cells that were released into the permissive temperature replicated and attained a 2C DNA content within 60 min after release (Fig. 5 B), whereas the cells placed at 11°C failed to replicate and remained with a 1C DNA content (Fig. 5A). This result suggests that execution of CDC45 function is dependent on CDC7. To determine whether the reverse might hold—i.e., that CDC7 function is dependent on execution of CDC45 function—we performed the reciprocal experiment. The cdc45-1 cdc7-4 cells were released from a cdc45 arrest at 11°C into media at either 38°C, the restrictive temperature for cdc7-4,or

FIG.5. CDC45 and CDC7 are dependent on one another for function. (A)Acdc45-1 cdc7-4 culture was arrested at 38°C, the restrictive temperature for cdc7-4. At 0 h, the culture was released into FIG. 4. Genomic footprinting of cdc45-1 cells. CDC45 and cdc45-1 medium at 11°C, the restrictive temperature for cdc45-1. Samples were cells were arrested at START with ␣-factor-containing medium at monitored by flow cytometry. (B) Cells were arrested in medium at 30°C (␣f), and synchronously released at 11°C in the presence of 38°C. At 0 min, the culture was released at 27°C, the permissive nocodazole for 9 h (11°C). Each sample was processed in duplicate for temperature for both cdc45-1 and cdc7-4.(C) Cells were arrested in genomic footprinting (A) and flow cytometry (B). The arrow indicates medium at 11°C. At 0 min, the culture was released at 38°C. (D) Cells the hypersensitive site which is present at the 2-micron origin during were arrested in medium at 11°C. At 0 min, the culture was released S, G2, and M phases of the cell cycle. at 27°C. Downloaded by guest on September 30, 2021 Genetics: Owens et al. Proc. Natl. Acad. Sci. USA 94 (1997) 12525

activity is regulated during the cell cycle, this regulation must occur by some mechanism besides modulation of protein levels or changes in subcellular localization. We note that it is formally possible that the epitope tag that we used to monitor Cdc45p levels affected the metabolism of the protein and thereby its steady-state levels. However, given that tagging the endogenous protein had no discernible effects on cell growth or replication, we anticipate that the levels of untagged Cdc45p will also prove to be constant once antibodies directed against Cdc45p itself become available. We have shown that Cdc45p executes a function that is dependent on passage through START and is essential for replication elongation. This conclusion is consistent with a role for CDC45 in replication initiation. Previous efforts to order these events using the semi-restrictive temperature of 15°C were complicated by an inability to fully block DNA replication and achieve a reversible cdc45-1 arrest (31). Shifting cdc45-1 cells to 15°C delayed and prolonged but did not prevent S phase. We were able to demonstrate a strict dependence of replication on CDC45 function because cdc45-1 achieves a tight, reversible arrest at 11°C. Moreover, at this fully restrictive temperature cdc45-1 cells were able to synthesize DNA with nearly wild-type kinetics when released from a HU arrest. FIG. 6. CDC45 and DBF4 are dependent on one another for This latter result is similar to observations reported at 15°C function. Experiments were performed identically to Fig. 5, except (31) and, with the caveat that only a single allele of cdc45 has dbf4-1 was used instead of cdc7-4. been examined, supports the notion that Cdc45p does not play an essential role in DNA elongation. 27°C, the permissive temperature for both mutants. The cells The events leading to the initiation of DNA replication can that were released into the permissive temperature were able be divided into at least two stages: assembly of pre-RCs at to replicate within 60 min (Fig. 5D), whereas the cells released origins shortly after mitosis and triggering of these complexes to 38°C did not replicate even after 120 min (Fig. 5C). As at the G1͞S boundary (16). Our data suggest that when additional controls we also repeated both temperature shift released from a START block at the restrictive temperature, experiments with cdc45-1 and cdc7-4 single mutants to confirm cdc45-1 cells failed to enter S phase and arrested with the that each is capable of releasing from their arrest when shifted pre-RC still present at the 2-micron origin. In contrast, wild- to the other temperature extreme. Both cdc45-1 cells released type cells triggered initiation, disassembled the pre-RC, and from an 11°C arrest into 38°C and cdc7-4 cells released from replicated their DNA. Given the correlation between the a 38°C arrest to 11°C were able to replicate normally (data not initiation of DNA replication and the disappearance of pre- shown). Thus, we conclude that Cdc45p and Cdc7p function RCs (16), the persistence of pre-RCs in cdc45-1-arrested cells interdependently—i.e., they are dependent on each other for suggests that most 2-micron origins have not fired. This function. observation argues for a role of Cdc45p in triggering the Given that Cdc7p kinase functions in association with a pre-RC. regulatory subunit, Dbf4p (12), we wondered whether Cdc45p The persistence of the pre-replicative genomic footprint in and Dbf4p share a similar interdependent relationship. Hence, cdc45-1 cells at the restrictive temperature raises the possibility we performed the identical reciprocal shift experiments in that Cdc45p is not a component of the pre-RC. However, we cdc45-1 dbf4-1 double mutants. Double mutant cdc45-1 dbf4-1 cannot rule out Cdc45p being a part of the pre-RC because the cells released from a dbf4-1 arrest at 38°C into the permissive incorporation of Cdc45p in the complex might not be affected temperature for both mutations, 27°C, replicated and attained by the cdc45-1 mutation. Furthermore, even if Cdc45p were a a 2C DNA content (Fig. 6B). In contrast, the same cells component of the pre-RC, its presence or absence in the released into 11°C failed to replicate and retained a 1C DNA complex may not affect the genomic footprint. content (Fig. 6A). This result suggests that execution of CDC45 Cdc7p kinase, in association with its regulatory subunit function is dependent on DBF4. Similarly, cdc45-1 dbf4-1 cells Dbf4p (12), acts at the last genetically defined cell cycle step released from a cdc45 arrest at 11°C into the permissive before entry into S phase (11) and is thought to help trigger temperature were able to replicate (Fig. 6D), whereas the cells initiation of DNA replication. Like the cdc45-1 mutant, cdc7 did not replicate when released into 38°C (Fig. 6C). This cells arrest in late G1 with the pre-RC still present at its origins finding indicates that execution of DBF4 function is dependent (16). Our demonstration that CDC45 functions interdepen- on CDC45. Thus, we conclude that, like CDC45 and CDC7, dently with both CDC7 and DBF4 further implicates CDC45 in CDC45 and DBF4 function interdependently. the triggering of initiation and suggests that Cdc45p acts in concert with Cdc7p͞Dbf4p kinase to carry out this event. DISCUSSION Genetic and biochemical studies suggests that the MCM family of proteins and ORC may also participate in the It has been demonstrated that CDC45 mRNA is periodically initiation events involving Cdc45p and Cdc7p͞Dbf4p. First, expressed during the cell cycle with levels peaking at the G1͞S cdc45-1 interacts genetically with mutants in several MCM and transition (32). This periodicity raises the possibility that ORC components (31–33), and Cdc45p physically associates CDC45 activity is regulated during the cell cycle through with at least one of the MCM proteins, Cdc46p͞Mcm5p (29). control of its protein levels. Although fluorescence microscopy Second, cdc7 and dbf4 deletion mutations can be suppressed by studies have demonstrated that CDC45 protein persists in the a mutation in CDC46͞MCM5 (30). Third, Cdc7p kinase can nucleus throughout the cell cycle (29), a quantitative deter- phosphorylate several MCM proteins in vitro (B. K. Tye and A. mination of protein levels had not been made. In this report, Sugino, personal communication). These observations raise we show by immunoblot analysis that CDC45 protein is several possibilities of what Cdc45p could be doing at the G1͞S maintained at constant levels in cycling cells. Thus, if Cdc45p boundary. One possibility is that it facilitates the activation of Downloaded by guest on September 30, 2021 12526 Genetics: Owens et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Cdc7p kinase in late G1. Another is based on the notion that 14. Dowell, S. J., Romanowski, P. & Diffley, J. F. (1994) Science 265, the MCM proteins and ORC are components of the pre-RC 1243–1246. (16, 23, 39) and that phosphorylation of these proteins by 15. Schwob, E., Bohm, T., Mendenhall, M. D. & Nasmyth, K. (1994) Cdc7p kinase contributes to activation of the pre-RC. In this Cell 79, 233–244. scenario, Cdc45p may regulate the activity of Cdc7p by con- 16. Diffley, J. F. X., Cocker, J. H., Dowell, S. J. & Rowley, A. (1994) trolling access of the kinase to its substrates in the pre-RC. Cell 78, 303–316. Finally, Cdc45p may itself be phosphorylated and activated by 17. Cocker, J. H., Piatti, S., Santocanale, C., Nasmyth, K. & Diffley, 379, Cdc7p͞Dbf4p to trigger the pre-RC. Elucidating the precise J. F. (1996) Nature (London) 180–182. 110, role of Cdc45p in triggering replication initiation will require 18. Detweiler, C. S. & Li, J. J. (1997) J. Cell Sci. 753–763. 19. Kearsey, S. E., Maiorano, D., Holmes, E. C. & Todorov, I. T. further biochemical investigation. (1996) BioEssays 18, 183–190. 20. Chong, J. P., Thommes, P. & Blow, J. J. (1996) Trends Biochem. We are grateful to David Botstein for the original cdc45-1 strain and Sci. 21, 102–106. Ross Okamura and Mary O’Riordan for technical assistance with flow 21. Tye, B.-K. (1994) Trends Cell Biol. 4, 160–166. cytometry and Southern blot analysis. We thank David Morgan, 22. Yan, H., Merchant, A. M. & Tye, B. K. (1993) Genes Dev. 7, Alexander Johnson, Erin O’Shea, and Ira Herskowitz for critically reading the manuscript and members of the Li lab for helpful 2149–2160. discussions. J.C.O. is supported by an Achievement Rewards for 23. Coleman, T. R., Carpenter, P. B. & Dunphy, W. G. (1996) Cell College Scientists Foundation graduate fellowship and by a National 87, 53–63. Institutes of Health training grant. C.S.D. is supported by a National 24. Muzi-Falconi, M., Brown, G. W. & Kelly, T. J. (1996) Curr. Biol. Defense Science and Engineering Grant from the Department of 6, 229–233. Defense and by a National Institutes of Health training grant. J.J.L. is 25. Wang, T. A. & Li, J. J. (1995) Curr. Opin. Cell Biol. 7, 414–420. a Lucille P. Markey Scholar, a Searle Scholar, and a Rita Allen 26. Diffley, J. F. (1996) Genes Dev. 10, 2819–2830. Foundation Scholar. 27. Epstein, C. B. & Cross, F. R. (1992) Genes Dev. 6, 1695–1706. 28. Schwob, E. & Nasmyth, K. (1993) Genes Dev. 7, 1160–1175. 1. Foss, M., McNally, F. J., Laurenson, P. & Rine, J. (1993) Science 29. Hopwood, B. & Dalton, S. (1996) Proc. Natl. Acad. Sci. USA 93, 262, 1838–1844. 12309–12314. 2. Fox, C. A., Loo, S., Dillin, A. & Rine, J. (1995) Genes Dev. 9, 30. Hardy, C. F., Dryga, O., Seematter, S., Pahl, P. M. & Sclafani, 911–924. R. A. (1997) Proc. Natl. Acad. Sci. USA 94, 3151–3155. 3. Bell, S. P., Kobayashi, R. & Stillman, B. (1993) Science 262, 31. Zou, L., Mitchell, J. & Stillman, B. (1997) Mol. Cell. Biol. 17, 1844–1849. 553–563. 4. Li, J. J. & Herskowitz, I. (1993) Science 262, 1870–1874. 32. Hardy, C. F. (1997) Gene 187, 239–246. 5. Loo, S., Fox, C. A., Rine, J., Kobayashi, R., Stillman, B. & Bell, 33. Hennessy, K. M., Lee, A., Chen, E. & Botstein, D. (1991) Genes S. (1995) Mol. Biol. Cell 6, 741–756. Dev. 5, 958–969. 6. Micklem, G., Rowley, A., Harwood, J., Nasmyth, K. & Diffley, 34. Guthrie, C. & Fink, G. R. (1991) Methods Enzymol. 194, 281–301. J. F. (1993) Nature (London) 366, 87–89. 35. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., 7. Dalton, S. & Whitbread, L. (1995) Proc. Natl. Acad. Sci. USA 92, Seidman, J. G., Smith, J. A. & Struhl, K. (1997) Current Protocols 2514–2518. 8. Hennessy, K. M., Clark, C. D. & Botstein, D. (1990) Genes Dev. in Molecular Biology (Wiley, New York). 4, 2252–2263. 36. Tyers, M., Tokiwa, G., Nash, R. & Futcher, B. (1992) EMBO J. 9. Whitbread, L. A. & Dalton, S. (1995) Gene 155, 113–117. 11, 1773–1784. 10. Yan, H., Gibson, S. & Tye, B. K. (1991) Genes Dev. 5, 944–957. 37. Diffley, J. F. X. & Cocker, J. H. (1992) Nature (London) 357, 11. Hartwell, L. H. (1976) J. Mol. Biol. 104, 803–817. 169–172. 12. Sclafani, R. A. & Jackson, A. L. (1994) Mol. Microbiol. 11, 38. Huibregtse, J. M. & Engelke, D. R. (1991) Methods Enzymol 194, 805–810. 550–562. 13. Jackson, A. L., Pahl, P. M., Harrison, K., Rosamond, J. & 39. Donovan, S., Harwood, J., Drury, L. S. & Diffley, J. F. (1997) Sclafani, R. A. (1993) Mol. Cell. Biol. 13, 2899–2908. Proc. Natl. Acad. Sci. USA 94, 5611–5616. Downloaded by guest on September 30, 2021