Regulatory mechanism of the initiation of DNA replication by cyclin-dependent kinase

In Sacchromyces cerevisiae, it is well known that bidirectional replication initiates at sequence specific locus on chromosome, called the autonomously replicating sequences (ARSs) (Campbell and Newlon, 1991). The initiation of DNA replication starts from formation of the origin recognition complex (ORC), which contains six subunits and binds directly to the origins of replicatuon. Then the ORC complex recruits two binding factors critical for the activation (Kelly and Brown, 2000), Cdc6 and Cdtl, which allow assembly of the mini-chromosome maintenance (MCM) complex onto the origins, completing assembly of the prereplicative complex (pre-RC) (Zou and Stillman, 2000; Jares and Blow, 2000;Walter and Newport, 2000). At the Gl/S transition, the pre-RC allows replication initiation by action of two protein kinases, S-phase cyclin-dependent kinase (S-Cdk; Cdc28) and Cdc7. Then Cdc45-Sld3, Dpb11-Sld2 and GINS complex associate with origins, dependently on the pre-RC (Takayama et al., 2003; Kubota et al., 2003), and DNA polymerases(α , δ and ε) are recruited to origins to initiate DNA synthesis. An essential protein for DNA replication, Cdc45, is known to be involved in initiation at origins and elongation of replication forks. A two-dimensional gel electrophoresis experiment has shown that a mutant cdc45-1, have a defect in the initiation step (Zou et al., 1997), while another mutant, heat-inducible degron cdc45-td cells, showed that Cdc45 plays an essential role in elongation as they cannot finish chromosome replication at the restrictive condition after early origin activation (Tercero et al., 2000). These observations suggest that Cdc45 could be a good candidate to be studied for elucidating how origin activation occurs and how elongation would be accomplished during DNA synthesis. In S.cerevisiae, Dpb11 has been shown to play a pivotal role in loading DNA polymerases onto replication origins. The inability of DPB11 mutants to restrain mitosis in the presence of incomplete replication suggests that Dpb11 is also needed for the replication checkpoint (Araki et al., 1995; Kamimura et al., 1998; Wang and Elledge, 1999). Dpb11 has four copies of the BRCT ( Brcal C- terminal) domain (Bork et al., 1997; Callebaut and Mornon, 1997; Zhang et al., 1998), and forms a complex with Pol ε and Sld2 (Masumoto et al., 2000; Kamimura et al., 1998; Wang and Elledge, 1999). BRCT domains are autonomously folding modules consisting of about 100 amino acids that were first recognized as a repeat in the C-terminus of the Breast Cancer Susceptibility gene l (BRCA1). BRCT domains are shown to bind proteins that have been phosphory\u27lated on either serine or threonine residues (Manke et al., 2003; Yu et al., 2003). Therefore, elucidation of the molecular mechanism in the interaction between BRCT domain and its physiological targets might be helpful to elucudate the cell cycle control including S phase regulation. The S1d2-Dpb11 complex is formed in vivo and is essential for chromosomal DNA replication (Kamimura et al., 1998). Sld2 has six closed matches to the preferred Cdk motif, S/T-P-X-K/R, S/T-P-K/R and K/R-S/T-P (X= any amino acid), and additional five S/T-P sites, which are clustered in 200-aa stretch (Pearson and Kemp, 1991). Masumoto et al showed that S1d2 is phosphorylated by S-Cdk and that this phosphorylation is necessary for the formaion of the Sld2-Dpb11 complex. Remarkably, the mutant allele of SLD2 that replaces all the serine or threonine residues in the preferred Cdk phosphorylation motif by alanine residues is inviable and is also defective in formation of Sld2-Dpb11 complex (2002). However, it is not well understood how the complex is formed between Sld2 and Dpb11. To understand the initiation of replication regulated by Cdks, Ms. Tak has studied two events in early S phase: stable association of Cdc45 with chromatin and association of Dpb11-Sld2 complex with origins. Although the association of Cdc45 with the pre-RC has been observed in even Gl phase by chromatin immuno-precipitation (ChIP) assay, its stable association to chromatin occurs only after activation of S-Cdks in Gl/S transition (Aparicio et al., 1999; Zou and Stillman, 2000; Kamimura et al., 2001). In Xenopus egg extract loading of Cdc45 onto chromatin in S phase is the last known step before origin unwinding and the commencement of DNA synthesis (Mimura et al., 2000). These results suggest that Cdc45 is limiting factor for the initiation of replication. While Dpb11 does not associate with origin without Cdk activity , Xenopus Cut5/Mus101 binds to chromatin in advance of Cdc45, rather than in a mutually dependent manner, but is needed for Cdc45 and polymerase α association steps (Hashimoto and Takisawa, 2003; Van Hatten et al., 2002). In this study, Ms. Tak isolated series of thermosensitive allele for CDC45 defective in the initiation of DNA replication. In thermosensitive alleles for SLD2 and DPB11, drcl-1 and dpb11-26, respectively, the chromatin binding assay revealed that the chromatinbound Cdc45 is significantly reduced even after Cdk activation. A mutant allele, cdc45-26, isolated in this study, abolishes association of Sld2-Dpb11 with the early-origins, but not affect the Sld2-Dpb11 complex formation. These results suggest that S-Cdk dependent stable association of Cdc45 and association of Dpb11-Sld2 complex with the origins are mutually dependent. Moreover Ms. Tak defined a Dpb11 binding region in Sld2 within a 39-amino acids stretch, partly overlapping with a cluster of the phosphorylation sites. The 39-aa peptide binds to C-terminal pair of BRCT domains in Dpb11 irrespective of phosphorylation. When MS. TAK fused 39-aa to a cluster of phosphorylation sites the interaction between the fragment of Sld2 and Dpb11 was phosphorylation dependent. Therefore, Ms. Tak propose that a cluster of phosphorylation sites in Sld2 regulates the affinity of 39-as binding domain to Dpb11. This model suggests an interesting possibility that Sld2 changes the conformation of Dpb11 to associate with other replication proteins, such as Sld3, Dpb2 and GINS