Purification and Characterization of theSchizosaccharomyces pombe Origin Recognition Complex

Abstract

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...

DNA replication origins in the Schizosaccharomyces pombe genome

“…Thus, the overall affinity of SpOrc4 for such a sequence would be a complex function of the number of possible binding modes and the binding affinity of each mode and would be expected to increase with AT content and length. Consistent with these ideas, it has been demonstrated experimentally that SpOrc4 has significant affinity for multiple sites within a single ars element (41)(42)(43)46). Moreover, genetic studies have shown that sequences capable of functioning as origins contain multiple redundant AT-rich elements that contribute to activity (14-16, 18, 38).…”

“…Biochemical studies have shown that the S. pombe origin recognition complex is targeted to origins by the N-terminal domain of SpOrc4, which contains nine AT-hook motifs (40)(41)(42)(43). The AT-hook motif binds to short AT tracts of 4-6 nt in length in the minor groove of DNA (44).…”

Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA

“…Furthermore, the spOrc4p alone binds to DNA as efficiently as spORC. The importance of the N-terminal AT hook domain was further substantiated by the finding that this region of spOrc4p is essential for viability in vivo (8).…”

“…The structure and initiator role of ORC is conserved in all eukaryotes, and six-subunit complexes have been purified from species including Schizosaccharomyces pombe (sp), Drosophila melanogaster (Dm), Xenopus laevis, and humans (7)(8)(9)(10)(11). In vitro and in vivo studies indicate that although ORC is essential for DNA replication in these species (1), the sequences recognized by these ORC complexes differ.…”

Noncanonical DNA Elements in the Lamin B2 Origin of DNA Replication

“…They could include (dT) n (dA) n sequences predominantly distributed in a mirror repeat manner, and as reported, involved in initiator protein binding (5,(12)(13)(14)(15)(16)(17)(18). The structural impact of these sequences was not investigated, but numerous other studies confirm that A and T tracts (depending on their distribution and size) could profoundly affect geometry of grooves, bending, and the ability to bend or formation of loops and multistranded structures (19).…”