The CENP-B homolog, Abp1, interacts with the initiation protein Cdc23 (MCM10) and is required for efficient DNA replication in fission yeast

DNA replication requires the assembly of replicative complexes (RCs) at chromosomal replication origins. The first step in this process involves assembly of pre-RCs in early G1 [1–6]. Formation of these complexes is restricted to a period of the cell cycle following conclusion of mitosis when c yclin-d ependent k inase (Cdk) activity is low [7–9]. Subsequent conversion of the pre-RC to an active replicative complex at the beginning of S phase is dependent on re-activation of Cdk activity that leads to the recruitment of essential replication proteins to origin DNA. This mechanism ensures that cells replicate once and only once during each cell division cycle [10]. Unlike simple organisms like bacteria, yeast requires that initiation of DNA replication occur on chromatin-bound templates. Although the details of how replication occurs on chromatin are poorly understood, it is possible that remodeling activities that promote protein-protein and protein-DNA interactions on chromatin are important to allow efficient replication of these templates [11–13].

Two proteins that are believed to be required for assembly of pre-RCs in fission yeast are Cdc18 (Cdc6) and Cdt1 (Cdt1) [14–18]. For clarity, all S. pombe gene/protein names will be followed by the corresponding S. cerevisiae gene/protein name in parentheses, where a clear homolog exists. These proteins facilitate the loading of Mini-Chromosome-Maintenance (MCMs) proteins to origin DNA in early G1 [19–21]. The Mcm2-7 complex is believed to function as the DNA replicative helicase that unwinds origin DNA at the start of S phase [22–24]. Two additional proteins, Sna41 (Cdc45) and Cdc23 (Mcm10) interact both physically and genetically with components of the initiation complex, and are believed to be important for recruitment of DNA polymerases to the origin-associated pre-RC [25–31].

The cdc23 (MCM10) mutant of S. pombe was originally identified as a cell cycle mutant defective in the completion of DNA synthesis and was later shown to block in early S phase [28, 32]. Subsequent cloning and characterization of the cdc23 (MCM10) gene demonstrated that it is homologous to S. cerevisiae MCM10, and was capable of rescuing the budding yeast mcm10 mutant, dna43-1 [33]. In S. cerevisiae, Mcm10 binds to replication origins during G1 and S phase suggesting it plays a critical role in both initiation and elongation of DNA replication [29]. However, in fission yeast it is not yet clear whether Cdc23 (Mcm10) binds origins in a cell cycle dependent manner [34]. More recently, Cdc23 (Mcm10) has been shown to interact directly with DNA polymerase α/primase and to stimulate primase activity in vitro [29, 35]. Cdc23 (Mcm10) has been shown to genetically and physically interact with components of the pre-RC, including Mcm2, 4, 5 and 6 and to facilitate chromatin binding of Cdc45 [28, 37]. Two-hybrid analysis also shows that Cdc23 (Mcm10) can interact with Orc1, 2, 5, and 6 [28, 36]. These data strongly support a role for Cdc23 (Mcm10) in the initiation of DNA replication. Also, biochemical analysis of purified Cdc23 (Mcm10) protein demonstrates that it is required for efficient phosphorylation of the Mcm2-7 complex by Dfp1-Hsk1 (Dbf4-Cdc7) kinase in vitro, and that Cdc23 (Mcm10) can directly interact with Dfp1-Hsk1 (Dbf4-Cdc7) [37].

A rs b inding p rotein 1 (Abp1) was first identified in a search for fission yeast proteins that could retard an ARS (A utonomously R eplicating S equence)-containing DNA fragment in a gel-shift mobility assay [38]. Although Abp1 was shown to be non-essential [39], the protein could bind very tightly to ARS elements in vitro. Independently, Abp1 was also identified as a protein that could bind centromeric DNA sequences [39]. These regions of DNA typically contain high concentrations of ARS-related sequences (Takahashi et al, 1992). Consistent with a role in either DNA replication or chromosome segregation deletion of abp1⁺ was shown to decrease mini-chromosome stability [39]. When carefully analyzed, many of the cells deleted for abp1⁺ displayed segregation defects suggesting that Abp1's primary function may be to ensure proper chromosome segregation at the conclusion of mitosis [39]. However, these observations do not rule out the possibility that Abp1 has a role in DNA replication. Moreover, two additional S. pombe Abp1-related proteins, called Cbh1 and Cbh2, have been identified and like Abp1, both were shown to be non-essential for viability [40–42]. However deletion of both Abp1 and Cbh1 lead to loss of viability and dramatic morphological changes, including branching and cell elongation. Therefore the function of these proteins is likely to be redundant and essential for normal cell cycle progression [42, 43]. More recently, Abp1 has been shown to bind directly to the outer repeats of the S. pombe centromere, promoting specific histone modifications that lead to the recruitment of Swi6 and gene silencing [44]. Although currently there is no evidence to suggest that either Abp1 or Swi6 interact with other regions of the genome apart from centromeres and telomeres, it is possible that they interact specifically with replication origin DNA and that their presence at these sites regulate initiation of DNA replication.

Using a two-hybrid system, we have identified Abp1 as a protein that interacts with Cdc23 (Mcm10). Genetic interactions between a cdc23 (mcm10) temperature-sensitive mutant and the Δabp1 strain provide further support that these two proteins functionally interact. We also show that deletion of abp1⁺ results in a delay in S phase when released from a G1 block consistent with Abp1 having a role in DNA replication initiation.

Initiation of DNA replication from chromatin-bound templates raises important questions concerning how DNA replication complexes gain access to origin DNA. It is likely that many auxiliary factors contribute to the assembly of both the pre-RC and its subsequent conversion to a fully functional replication fork on chromatin. Yeast genetics has provided a powerful tool to identify some of the key proteins that are required for assembly of these complexes. One of these proteins, called Cdc23 (Mcm10), is conserved from yeast to man, and is required for the early events of DNA replication initiation. It has been shown to interact directly with DNA polymerase alpha/primase and to stimulate primase activity in vitro. However, its precise role in S phase is still unclear. In an attempt to gain some insights into the function of Cdc23, we conducted a two-hybrid screen for Cdc23-interacting proteins. One of the proteins identified using this screen is Abp1, a protein that had previously been shown to bind to both ARS elements and centromeric DNA sequences. Although its primary function is thought be in chromosome segregation, we show that Abp1 may also have an additional role in DNA replication initiation.

First, Abp1 not only interacts with Cdc23 (Mcm10) protein in the two-hybrid analysis, but deletion of Abp1 lowers the restrictive temperature for cdc23-M36, consistent with its proposed role in DNA replication. Although two other proteins show significant homology to Abp1, deletion of either of these does not alter the phenotype of the cdc23 temperature-sensitive mutant, suggesting that Cdc23 (Mcm10) specifically interacts with Abp1. Furthermore, a more extensive genetic analysis of double mutants between Δabp1 and other replication mutants reveals that Δabp1 genetically interacts with both orc2-7 and cdc30-2H4 (encoding a mutant form of Orc1 and Orc2, respectively) and cdc18-K46 (CDC6) and cdc21-M68 (MCM4) (two mutants that are defective in the assembly of the pre-RC) providing additional evidence that Abp1 interacts with components of the replication initiation complex.

Our flow cytometry analysis suggests that cdc23⁺ (MCM10) is required for DNA replication initiation. Previous studies also indicate that following a shift to the restrictive temperature of 36°C, cdc23 temperature-sensitive mutant cells arrest in early-S phase [32]. In our experiments a shift to the semi-permissive temperature of 30°C causes accumulation of cells with what appears to be a near 1C or G1-like DNA content suggesting that Cdc23, like its counterpart Mcm10 in budding yeast, is required for DNA replication initiation. This confirms that Abp1 interacts with a bona-fide DNA replication initiation protein.

Finally, we demonstrate, that cells deleted for Abp1 are significantly delayed from entering S phase following release from a G1 block. This delay is only observed following arrest in G1, and is not observed following release from an S phase arrest imposed by hydroxyurea. This implies that loss of Abp1 during S phase has no effect on replication kinetics, whilst loss of Abp1 prior to S phase can impede initiation.

It is still not clear how Abp1 might function to facilitate DNA replication initiation. One possibility is that Abp1, together with other chromatin-bound proteins, is important to remodel replication origins to allow access to the replication machinery. Alternatively, Abp1 might have a specific function in regulating origin firing in centromeric repeats. Interestingly, Abp1 has been shown to be important for heterochromatin modifications that are required for recruitment of Swi6 to chromatin where Swi6 is then responsible for nucleating formation of silent chromatin. So at least in this case, the presence of Abp1 can directly influence chromatin structure in relationship to DNA transcription. Note that the outer repeat region of the centromere where Abp1 binds was shown to be rich in ARS elements, and the ARSs were indeed functional as early replication origins [45]. It will be interesting to determine if Abp1, perhaps in conjunction with particular histone modifications and Swi6, may play a similar role in DNA replication.