Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 May;20(9):3086-96.
doi: 10.1128/mcb.20.9.3086-3096.2000.

Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase

Affiliations
Free PMC article

Assembly of a Complex Containing Cdc45p, Replication Protein A, and Mcm2p at Replication Origins Controlled by S-phase Cyclin-Dependent Kinases and Cdc7p-Dbf4p Kinase

L Zou et al. Mol Cell Biol. .
Free PMC article

Abstract

In Saccharomyces cerevisiae, replication origins are activated with characteristic timing during S phase. S-phase cyclin-dependent kinases (S-CDKs) and Cdc7p-Dbf4p kinase are required for origin activation throughout S phase. The activation of S-CDKs leads to association of Cdc45p with chromatin, raising the possibility that Cdc45p defines the assembly of a new complex at each origin. Here we show that both Cdc45p and replication protein A (RPA) bind to Mcm2p at the G(1)-S transition in an S-CDK-dependent manner. During S phase, Cdc45p associates with different replication origins at specific times. The origin associations of Cdc45p and RPA are mutually dependent, and both S-CDKs and Cdc7p-Dbf4p are required for efficient binding of Cdc45p to origins. These findings suggest that S-CDKs and Cdc7p-Dbf4p promote loading of Cdc45p and RPA onto a preformed prereplication complex at each origin with preprogrammed timing. The ARS1 association of Mcm2p, but not that of the origin recognition complex, is diminished by disruption of the B2 element of ARS1, a potential origin DNA-unwinding element. Cdc45p is required for recruiting DNA polymerase alpha onto chromatin, and it associates with Mcm2p, RPA, and DNA polymerase epsilon only during S phase. These results suggest that the complex containing Cdc45p, RPA, and MCMs is involved in origin unwinding and assembly of replication forks at each origin.

Figures

FIG. 1
FIG. 1
Cdc45p associates with Mcm2p, Polɛ, and RPA p70 in vivo. (A) Mcm2p, PolɛHA3p, and RPA p70 coprecipitate specifically with Cdc45myc3p. Cells expressing (+) both PolɛHA3p and Cdc45myc3p (YB0550) or only PolɛHA3p (OAY618) were arrested in S phase with 0.1 M HU for 2 h. Whole-cell extracts were prepared and subjected to immunoprecipitations (IP) with non-cross-linked anti-myc antibody. ∗, dimers of immunoglobulin G. (B) Mcm2p, PolɛHA3p and Cdc45myc3p coprecipitate specifically with RPA p70. Whole-cell extract was prepared from S-phase wild-type (YB0550) cells. Immunoprecipitations were performed with either normal rabbit serum (NRS) or anti-RPA p70 antibody. (C) RPA p70 coprecipitates with Mcm2p only in wild-type but not in mcm2-1 cell extracts. Wild-type (YB0550) (WT) and mcm2-1 (YB0551) cells were arrested with HU for 2 h at the indicated temperatures. Immunoprecipitations were carried out with anti-Mcm2p antibody cross-linked to protein G. (D) Cdc45HA3p coprecipitates with Mcm2p only in wild-type but not in mcm2-1 cell extracts. Wild-type (YB0469) and mcm2-1 (YB0476) cells expressing Cdc45HA3p were arrested as in panel C, and immunoprecipitations were performed with non-cross-linked anti-Mcm2p antibody. Shown are immunoblots of the proteins present in the immunoprecipitation described above. The immunoblotting antibodies were against the proteins indicated at the right of each panel.
FIG. 2
FIG. 2
Cdc45p interacts with Mcm2p, Polɛ, RPA p70, and p34 in a cell cycle-dependent manner. (A) Immunoprecipitation (IP) of Cdc45myc3p across the cell cycle. Wild-type cells expressing both PolɛHA3p and Cdc45myc3p (YB0550) were synchronized in G1 with α-factor and then released into yeast-peptone-dextrose medium at 25°C. The cells were collected at the indicated time points. Whole-cell extracts were prepared and subjected to immunoprecipitations with anti-myc antibody. (B) DNA content of the time point samples used in panel A.
FIG. 3
FIG. 3
S-CDK activity is required for the interactions among Cdc45p, Mcm2p, and RPA. (A) Overexpression of Sic1ΔNTp prevents binding of Cdc45p to Mcm2p. Cells bearing GAL1-SIC1ΔNT (YB0553) were arrested with α-factor in raffinose (raf)-containing medium and then released into medium containing either raffinose or galactose (gal) at 25°C. As a control, wild-type (WT) cells (YB0469) that do not carry GAL1-SIC1ΔNT were also synchronized with α-factor in raffinose and released into galactose. Whole-cell extracts were prepared at the indicated time points and were immunoprecipitated with anti-Mcm2p antibody. Cdc45HA3p coprecipitated with Mcm2p was analyzed by immunoblotting. (B) Overexpression of Sic1ΔNTp prevents binding of RPA p70 and p34 to Mcm2p. RPA p70 and p34 coprecipitated with Mcm2p in panel A were analyzed by immunoblotting. (C) DNA content of the samples used in panels A and B.
FIG. 4
FIG. 4
Cell cycle regulation of association of Cdc45p with ARS-containing fragments. (A) Cdc45p and Mcm2p associate with origins differently in G1 and early S phase. Wild-type (WT) cells expressing Cdc45HA3p (YB0469) were arrested in G1 with α-factor and then released into yeast-peptone-dextrose medium at 25°C. The cells were collected at the α-factor block or 30 min after release. CHIP analyses of Mcm2p and Cdc45p were performed in parallel. WCE, input DNA prepared from the whole-cell extract; ARS1-4kb, a region 4 kb away from ARS1 towards the left telomere. (B) Association of Cdc45p with different origins during the cell cycle. Wild-type cells were synchronized in G1 with α-factor and then released into YPD at 25°C. CHIP analysis of Cdc45p was performed at each time point. +4 kb and −8 kb, regions on both sides of ARS1 that are 4 or 8 kb away. (C) DNA content of the time point samples used in panels B and D. (D) Association of Cdc45p with ARS1 is delayed in the absence of CLB5 and CLB6. YB0477 (clb5,6Δ and CDC45HA3) cells were released from an α-factor block at 25°C. CHIP analysis of Cdc45p was performed as described above.
FIG. 5
FIG. 5
Associations of Cdc45p with chromatin, Mcm2p, and ARS1 are affected in cdc7 and dbf4 mutants. (A) Association of Cdc45p with chromatin is reduced in cdc7 and dbf4 mutants. Wild-type (YB0469) (WT), cdc7-1 (YB0472), cdc7-4 (YB0547), and dbf4-1 (YB0548) cells expressing Cdc45HA3p were synchronized with α-factor and released at either 25 or 35°C. The cells were collected at the α-factor block or 30 min after release. Lysates were prepared and subjected to chromatin fractionation. Immunoblots of Cdc45p and Orc3p in the chromatin sediments (P) and Cdc45p in the supernatants (S) are shown. (B) Cdc45p-Mcm2p interaction is reduced in cdc7 and dbf4 mutants at the nonpermissive temperature. Cells were arrested as in panel A and released at 37°C. Whole-cell extracts (WCE) were prepared from the cells collected at the α-factor block or 30 min after release and subjected to immunoprecipitations (IP) using an anti-Mcm2p antibody. (C) Association of Cdc45p with ARS1 is undetectable in cdc7-1, cdc7-4, and dbf4-1 cells. Wild-type and mutant cells were synchronized with α-factor as in panel A and released at 35°C. The cells were collected at the indicated time points and analyzed by Cdc45p CHIP. (D) DNA content of the samples used in panel C.
FIG. 6
FIG. 6
Origin associations of Cdc45p and RPA are mutually dependent. (A) ARS1 association of RPA p70 requires Cdc45p. Wild-type (WT) and cdc45-1 (YB0298) cells were synchronized in G1 with α-factor and released into medium containing 0.1 M HU at 13°C. CHIP analysis of RPA p70 was performed at the indicated time points. (B) DNA content of the cells blocked and released as in panel A but in the absence of HU. (C) ARS1 association of Cdc45p requires RPA p34. Wild-type and rfa2-2 (YB0549) cells were synchronized in G1 with α-factor and released at 35°C in the absence of HU. CHIP analysis of Cdc45p was performed at the indicated time points. (D) DNA content of the samples used in panel C. WCE, input DNA from whole-cell extract.
FIG. 7
FIG. 7
Cdc45p is required for loading Polα p86 but not Dbf4p onto chromatin. (A) Wild-type (K6388) (WT) and cdc45-1 (YB0552) cells were synchronized in G1 with α-factor and released at 13°C. Cells were collected at the indicated time points and were processed for chromatin fractionation. The chromatin-sediment fractions (P) were analyzed by immunoblotting. (B) DNA content of the samples used in panel A.
FIG. 8
FIG. 8
The B2 element of ARS1 is involved in association with Mcm2p but not ORC. The Cdc45HA3p-expressing cells with the chromosomal ARS1 mutated in either the A (YB0576), B1 (YB0577), B2 (YB0578), or B3 (YB0579) element were analyzed by CHIP assays. Asynchronously growing wild-type (WT) and ARS1 mutant cells were subjected to CHIP analysis with antibodies against Orc2p-Orc3p (antibodies against Orc2p and Orc3p were mixed and used together) and Mcm2p, respectively.

Similar articles

See all similar articles

Cited by 137 articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback