Identification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe

The origin recognition complex (ORC), first identified in Saccharomyces cer evisiae (sc), is a six-subunit protein complex that binds to DNA origins. H ere, we report the identification and cloning of cDNAs encoding the six sub units of the ORC of Schizosaccharomyces pombe (sp). Sequence analyses revea led that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts from S. cerevisiae, Xenopus, Drosophila, and human. In contra st, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Ch uang and Kelly [(1999) Proc. Natl. Acad. Sci. USA 96, 2656-2661], the C-ter minal region of spOrc4 is also conserved whereas the N terminus uniquely co ntains repeats of a sequence that binds strongly to AT-rich DNA regions. Co nsistent with this, extraction of 5. pombe chromatin with 1 M NaCl, or afte r DNase I treatment, yielded the six-subunit ORC, whereas extraction with 0 .3 M resulted in five-subunit ORC lacking spOrc4p. The spORC can be reconst ituted in vitro with all six recombinant subunits expressed in the rabbit r eticulocyte system. The association of spOrc4p with the other subunits requ ired the removal of DNA from reaction mixture by DNase I. This suggests tha t a strong interaction between spOrc4p and DNA can prevent the isolation of the six-subunit ORC. The unique DNA-binding properties of the spORC may co ntribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication in S. pombe.