The origin recognition complex (ORC) plays a central role in the initiation of DNA replication in eukaryotic cells. It interacts with origins of DNA replication in chromosomal DNA and recruits additional replication proteins to form functional initiation complexes. These processes have not been well characterized at the biochemical level except in the case of Saccharomyces cerevisiae ORC. We report here the expression, purification, and initial characterization of Schizosaccharomyces pombe ORC (SpORC) containing six recombinant subunits. Purified SpORC binds efficiently to the ars1 origin of DNA replication via the essential Nterminal domain of the SpOrc4 subunit which contains nine AT-hook motifs. Competition binding experiments demonstrated that SpORC binds preferentially to DNA molecules rich in AT-tracts, but does not otherwise exhibit a high degree of sequence specificity. The complex is capable of binding to multiple sites within the ars1 origin of DNA replication with similar affinities, indicating that the sequence requirements for origin recognition in S. pombe are significantly less stringent than in S. cerevisiae. We have also demonstrated that SpORC interacts directly with Cdc18p, an essential fission yeast initiation protein, and recruits it to the ars1 origin in vitro. Recruitment of Cdc18p to chromosomal origins is a likely early step in the initiation of DNA replication in vivo. These data indicate that the purified recombinant SpORC retains at least two of its primary biological functions and that it will be useful for the eventual reconstitution of the initiation reaction with purified proteins.In bacteria, bacteriophage, and animal viruses the initiation of DNA replication takes place at defined nucleotide sequences known as origins of replication (1). Initiator proteins bind to such origin sequences and promote the biochemical steps leading to the establishment of replication forks. The interaction of initiator proteins with origins is less well understood in the case of eukaryotic cells where initiation of DNA replication occurs at multiple sites along chromosomal DNA (2). The best characterized eukaryotic chromosomal origins of replication are those of the budding yeast Saccharomyces cerevisiae. Like the origins of prokaryotes and animal viruses, budding yeast origins are modular in nature and are composed of several short, well defined sequence blocks distributed over a region of ϳ100 -150 bp (3-5). The most highly conserved sequence block of budding yeast origins is the A domain which contains an essential 11-bp ARS consensus sequence. An additional, less well conserved sequence block, referred to as the B domain, serves to enhance the efficiency of origin utilization (3-5). The six-subunit S. cerevisiae Origin Recognition Complex (ScORC) 1 binds specifically to the ARS consensus sequence in a reaction requiring ATP (6). Genetic and biochemical studies have established that ORC plays a central role in the initiation of DNA replication and that it functions, at least in part, to recruit es...

Heterochromatin in S. pombe is associated with gene silencing at telomeres, the mating locus and centromeres. The compact heterochromatin structure raises the question how it unpacks and reforms during DNA replication. We show that the essential DNA replication factor Cdc18 (CDC6) associates with heterochromatin protein 1 (Swi6) in vivo and in vitro. Biochemical mapping and mutational analysis of the association domains show that the N-terminus of Cdc18 interacts with the chromoshadow domain of Swi6. Mutations in Swi6 that disrupt this interaction disrupt silencing and delay replication in the centromere. A mutation cdc18-I43A that reduces Cdc18 association with Swi6 has no silencing defect at the centromere, but changes Swi6 distribution and accelerates the timing of centromere replication. We suggest that fine tuning of Swi6 association at replication origins is important for negative as well as positive control of replication initiation.

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