Ordered assembly of Sld3, GINS and Cdc45 is distinctly regulated by DDK and CDK for activation of replication origins

Abstract

Initiation of chromosome DNA replication in eukaryotes is tightly regulated through assembly of replication factors at replication origins. Here, we investigated dependence of the assembly of the initiation complex on particular factors using temperature-sensitive fission yeast mutants. The psf3-1 mutant, a GINS component mutant, arrested with unreplicated DNA at the restrictive temperature and the DNA content gradually increased, suggesting a defect in DNA replication. The mutation impaired GINS complex formation, as shown by pull-down experiments. Chromatin immunoprecipitation assays indicated that GINS integrity was required for origin loading of Psf2, Cut5 and Cdc45, but not Sld3. In contrast, loading of Psf2 onto origins depended on Sld3 and Cut5 but not on Cdc45. These results suggest that Sld3 functions furthest upstream in initiation complex assembly, followed by GINS and Cut5, then Cdc45. Consistent with this conclusion, Cdc7-Dbf4 kinase (DDK) but not cyclin-dependent kinase (CDK) was required for Sld3 loading, whereas recruitment of the other factors depended on both kinases. These results suggest that DDK and CDK regulate distinct steps in activation of replication origins in fission yeast.

Figure 1

Defective chromosome replication owing to the absence of an intact GINS complex in the fission yeast psf3 mutant. (A) Wild-type and psf3-1 cells grown at 25°C in EMM medium were incubated at 36°C for the indicated time and DNA content was analyzed by flow cytometry. Positions of 1C and 2C DNA contents are indicated. (B) Untagged (−tag, lanes 1 and 6), psf2-flag (wt, lanes 2, 3 and 7, 8) and psf2-flag psf3-1 (psf3-1, lanes 4, 5 and 9, 10) cells were grown at a permissive temperature (25°C) and portions were shifted to the restrictive temperature (36°C) for 2 h. Proteins in whole-cell extracts (WCE, lanes 1–5) and four times more amount of immunoprecipitates with anti-FLAG antibody (FL-IP, lanes 6–10) were separated by 15% polyacrylamide gel electrophoresis and analyzed by Western blotting with anti-FLAG, Psf1, Psf3 and Sld5 antibodies. Positions of the expected molecular masses for Psf2-FLAG, Psf1, Psf3 and Sld5 along with those for molecular weight markers are indicated. The bands indicated by asterisks on anti-FLAG and anti-Psf1 staining are IgG light chain and a nonspecific protein, respectively.

Figure 2

Origin association of GINS is impaired by psf3 mutation. (A) The experimental scheme is shown. psf2-flag nda3-KM311 (wild type) and psf3-1 psf2-flag nda3-KM311 (psf3-1) cells were arrested at metaphase by incubation at 20°C for 4 h and then shifted to 37°C (time 0), with addition of hydroxyurea (HU, 15 mM) in the case of wild type. Aliquots of cultures taken at the indicated time points were analyzed by flow cytometry and ChIP assay. (B) DNA contents of wild type with HU and psf3-1 without HU were analyzed by flow cytometry. (C) Positions of ars2004 and non-ars1 fragments, amplified by PCR in ChIP assay, are shown on the relevant portion of chromosome II. The distance (kb) from the left end of the chromosome and the length of the amplified fragment (bp) are indicated. (D) Immunoprecipitated DNA from wild-type extracts with anti-Mcm6 (upper panel) or with Psf2-FLAG (lower panel) at the indicated time points was used as template for PCR with ars2004 (upper bands) and non-ars1 (lower bands) primers. PCR from the total cellular DNA prepared without IP yielded similar amplification of two fragments (lane W). (E) DNA immunoprecipitated from psf3-1 cell extracts was analyzed as in (D).

Figure 3

Genetic interactions of psf3 with initiation complex components. (A) Gene-dosage suppression of the temperature sensitivity of psf3-1. Aliquots of leu1-32 psf3-1 cells harboring the p940 vector alone, p940-sld5⁺, p940-psf1⁺, p940-spf2⁺, p940-psf3⁺, p940-sld3⁺ and p940-drc1⁺ were spotted on EMM plates after 10-fold serial dilution and incubated at permissive (25°C) and restrictive (36°C) temperatures. (B) Growth of double mutants. Ten-fold serial dilutions of indicated genotype cells were spotted onto YE plates followed by incubation at the indicated temperatures. The alleles used were psf3-1, cut5-T401, cdc20-P7 (Polɛ) and pol1-1 (Polα). (C) Summary of growth of double mutants. Growth of double mutants was examined as in (B). The plus sign shows growth as good as wild type and the minus and plus/minus signs indicate the absence of growth and reduced number of colonies, respectively.

Figure 4

Localization of Sld3 but not Cdc45, Cut5, Drc1 or Dpb2 at replication origin in psf3-1 mutant. Aliquots of nda3-KM311 (A, wild type) and psf3-1 nda3-KM311 (B, psf3-1) derivatives carrying sld3-flag, cdc45-flag, cut5-flag, drc1-flag or dpb2-flag were arrested at metaphase by culturing at 20°C for 4 h and then incubated at 37°C. Hydroxyurea (15 mM) was added to wild-type cells upon the shift to 37°C. Immunoprecipitated DNA with anti-Mcm6, anti-FLAG or anti-Rpa2 antibody was analyzed by PCR with the ars2004 (upper bands) and non-ars1 (lower bands) primers. PCR products from total DNA without IP are shown in lane W.

Figure 5

Origin loading of GINS depends on Sld3 and Cut5 but not Cdc45. (A) DNA contents of sld3-10 psf2-flag nda3-KM311 (sld3), sna41-928/cdc45 psf2-flag nda3-KM311 (cdc45) and cut5-T401 psf2-flag nda3-KM311 (cut5) cells released from metaphase block were analyzed by flow cytometry. ChIP assays were carried out with anti-Mcm6 and anti-FLAG antibodies from sld3 (B) and cut5 (C), with anti-Mcm6, anti-FLAG and anti-Rpa2 antibodies from cdc45 (D), as described in Figure 2, using ars2004 (upper bands) and non-ars1 (lower bands) primers. PCR products from total cellular DNA without IP are shown in lane W. (E) Association of Mcm6 and Cut5-FLAG with origin in sna41-928/cdc45 cut5-flag nda3-KM311 cells after release from metaphase block was analyzed by ChIP assay.

Figure 6

Origin association of Sld3 depends on DDK but not on CDK. Aliquots of nda3-KM311 sld3-flag cdc45-myc and nda3-KM311 psf2-flag cells (wild type) were released from metaphase block at 36°C, as described in Figure 2. DNA contents of nda3-KM311 sld3-flag cdc45-myc (wild type), hsk1-89 nda3-KM311 sld3-flag cdc45-myc (hsk1) and cdc2-33 nda3-KM311 sld3-flag cdc45-myc (cdc2) after release at 36°C from metaphase block, as described in Figure 2, were analyzed by flow cytometry (A). Localization of Mcm6, Sld3-FLAG, Cdc45-Myc and Psf2-FLAG at ars2004 in wild type (B), hsk1 (C) and (C) cdc2 (D) derivatives was analyzed by ChIP assays. Three primer sets, non-arsA (upper bands), ars2004 (middle bands) and non-arsB (lower bands), were used for PCR (Ogawa et al, 1999). PCR products without IP are shown in lane W.

Figure 7

A model for ordered formation of initiation complex in fission yeast. (A) Based on dependence among Sld3, GINS, Cut5 and Cdc45 deduced from this study, a model for ordered assembly of replication factors at replication origins in fission yeast is presented. At the onset of S phase, Sld3 binds to replication origins independent of association of GINS, Cut5 or Cdc45 (Step 1). This step requires DDK function. Depending on Sld3 and CDK, GINS and Cut5 then bind to origins in a mutually dependent manner (Step 2). Finally, Cdc45 is recruited depending on all of Sld3, GINS, DDK and CDK (Step 3). At late-firing origins, association of Sld3 with Cut5 and Cdc45 may occur before their origin loading. (B) A model for assembly of replication factors on the early replication origins in budding yeast is presented. In G1 phase arrested by α-factor, Sld3 and Cdc45 associate with the early origins (Step 1). Upon activation of CDK and DDK at the beginning of S phase, Dpb11, Sld2 and GINS are recruited to early origins, in a mutually dependent manner, and Cdc45 becomes stably associated with the origins (Step 2).