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Molecular and Cellular Biology, December 2001, p. 8095-8103, Vol. 21, No. 23
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.23.8095-8103.2001

Site-Specific DNA Binding of the Schizosaccharomyces pombe Origin Recognition Complex Is Determined by the Orc4 Subunit

Daochun Kong and Melvin L. DePamphilis*

National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892-2753

Received 9 July 2001/Returned for modification 22 August 2001/Accepted 31 August 2001

The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. 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 of 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 cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit.

* Corresponding author. Mailing address: National Institute of Child Health and Human Development, Building 6, Room 416, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-2753. Phone: (301) 402-8234. Fax: (301) 480-9354. E-mail: depamphm{at}mail.nih.gov.

Molecular and Cellular Biology, December 2001, p. 8095-8103, Vol. 21, No. 23
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.23.8095-8103.2001


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