Sld3, which interacts with Cdc45 (Sld4), functions for chromosomal DNA replication in Saccharomyces cerevisiae

Abstract

Cdc45, which binds to the minichromosomal maintenance (Mcm) proteins, has a pivotal role in the initiation and elongation steps of chromosomal DNA replication in eukaryotes. Here we show that throughout the cell cycle in Saccharomyces cerevisiae, Cdc45 forms a complex with a novel factor, Sld3. Consistently, Sld3 and Cdc45 associate simultaneously with replication origins in the chromatin immunoprecipitation assay: both proteins associate with early-firing origins in G(1) phase and with late-firing origins in late S phase. Moreover, the origin associations of Sld3 and Cdc45 are mutually dependent. The temperature-sensitive sld3 mutation confers a defect in DNA replication at the restrictive temperature and reduces an interaction not only between Sld3 and Cdc45, but also between Cdc45 and Mcm2. These results suggest that the Sld3-Cdc45 complex associates with replication origins through Mcm proteins. At the restrictive temperature in sld3-5 cells, replication factor A, a single-strand DNA binding protein, does not associate with origins. Therefore, the origin association of Sld3-Cdc45 complex is prerequisite for origin unwinding in the initiation of DNA replication.

Activation of the DNA Damage Checkpoint in Mutants Defective in DNA Replication Initiation

“…In addition to the previously mentioned Orc, Cdc18, Cdt1, and Mcm proteins, they include the Gins complex (Alberghina et al, 1983;Takayama et al, 2003;Gambus et al, 2006;Labib and Gambus, 2007), Sna41/Cdc45 (Miyake and Yamashita, 1998;Uchiyama et al, 2001a,b), Sld2, and Sld3 (Kamimura et al, 1998(Kamimura et al, , 2001Nakajima and Masukata, 2002;Kanemaki and Labib, 2006;Tanaka et al, 2007;Zegerman and Diffley, 2007). In addition, our studies on DNA polymerase epsilon have suggested that along with its presumed function in DNA replication elongation, the C-terminal half of this protein is required for assembly of the DNA replication initiation complex (Feng and D'Urso, 2001;Feng et al, 2003).…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…A physical association between the two proteins has been suggested by co-immunoprecipitation after chemical crosslinking and ChIP experiments indicate that both proteins associate with the origins at the same time. (81) The mutant allele sld3-5 could not initiate DNA replication from the ARS1 origin, as determined by two-dimensional gel analysis. (81) SLD5 encodes a 35 kDa protein essential for cell growth.…”

“…(81) The mutant allele sld3-5 could not initiate DNA replication from the ARS1 origin, as determined by two-dimensional gel analysis. (81) SLD5 encodes a 35 kDa protein essential for cell growth. A combination of genetic and biochemical assays led to the identification of three Sld5-interacting proteins, PSF-1, PSF-2 and PSF-3 (''partners of Sld-five'').…”

Mcm10 interacts with Rad4/Cut5TopBP1 and its association with origins of DNA replication is dependent on Rad4/Cut5TopBP1

“…Cdc45 recruitment appears to require direct interaction with Mcm complex and the mcm5-bob1-1 mutation results in DDK-independent loading of Cdc45 and the bypass of the DDK requirement for initiation of DNA replication (13,14,15). Cdc45 loading 5 exhibits interdependence with Sld3, which in the light of the data discussed above, offers a compelling mechanism for combining the CDK and DDK activities in the initiation of DNA replication (3). Sld3 interacts with and is required for loading of the GINS complex in budding yeast (16).…”

“…Formation of this pre-replication complex (pre-RC) prior to S phase licenses origins for replication, but initiation of DNA replication requires the activity of two protein kinases, CDK (cyclin dependent kinase) and DDK (Dbf4 dependent kinase), and the assembly of other factors including Cdc45, GINS, Mcm10, Sld2 and Sld3. In budding yeast Cdc45 and Sld3 associate with early firing origins in G1, prior to CDK and DDK activity, and with late firing origins in S phase (3,4). Evidence from budding yeast has demonstrated that Sld2 and Sld3 recruitment are pivotal events in replication initiation at origins and the two proteins are critical targets of the S phase CDK (5,6,7,8).…”