Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12521–12526, November 1997 Genetics CDC45 is required in conjunction with CDC7yDBF4 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 Clb5pyClb6p 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 Cdc7pyDbf4p, a (16). This transition presumably reflects the simultaneous kinase implicated in the G1yS 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 CDC7yDBF4 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 G1yS 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 Cdc7pyDbf4p 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 mainte- nance of the pre-RC (17, 18). Other candidates include the six Media and Budding Index. Yeast extractypeptone (YEP) members of the MCM family of proteins (MCM2, MCM3, medium (34) was supplemented with 2% dextrose (YEPD). MCM5yCDC46, 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, a-factor was used at 50 ngyml (for bar1 cells) or 10 mgyml 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, minichro- mosome 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 0027-8424y97y9412521-6$2.00y0 Microbiology, Box 0414, University of California, San Francisco, CA PNAS is available online at http:yywww.pnas.org. 94143-0414. e-mail: [email protected]. 12521 Downloaded by guest on September 30, 2021 12522 Genetics: Owens et al. Proc. Natl. Acad. Sci. USA 94 (1997) 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 G1yS 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, MATa 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 a-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 59-GGCGGCCGCGCACCGGTG-39 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 59 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.