The Schizosaccharomyces Pombe Origin Recognition Complex Interacts with Multiple AT-rich Regions of the Replication Origin DNA by Means of the AT-hook Domains of the SpOrc4 Protein

The interaction between an origin sequence and the origin recognition complex (ORC), which is highly conserved in eukaryotes, is critical for the initiation of DNA replication. In this report, we have examined the interaction between the Schizosaccharomyces pombe (sp) autonomously replicating sequence 1 (ars1) and the spORC. For this purpose, we have purified the spORC containing all six subunits, a six-subunit complex containing the N-terminal-deleted spOrc4 subunit (spORC(Delta N-Orc4)), and the spOrc4 subunit by using the baculovirus expression system. Wild-type spORC showed sequence-specific binding to ars1, and the spOrc4 protein alone showed the same DNA-binding properties as wild-type spORC. In contrast, the spORC(Delta N-Orc4) and the Delta N-spOrc4p alone did not bind significantly to ars1. These findings indicate that the N-terminal domain of the spOrc4 protein that contains multiple AT-hook motifs is essential for the ars1-binding activity. DNA-binding competition assays with fragments of ars1 and DNase I footprinting studies with full-length ars1 revealed that the spORC interacted with several AT-rich sequence regions of ars1. These DNA-binding properties of spORC correlate with the previously determined sequence requirements of the S. pombe ars1. These studies indicate that because of its unique Orc4 subunit, S. pombe uses a mechanism to recognize its origins different from that used by Saccharomyces cerevisiae.