Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit

The mechanism by which origin recognition complexes (ORCs) identify replica tion origins was investigated using purified Orc proteins from Schizosaccha romyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origi n activity. These sites consisted of clusters of A or T residues on one str and but were devoid of either alternating A and T residues or GC-rich seque nces. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection o f specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the ce ll division cycle. These results reveal that the S. pombe ORC binds to mult iple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.