Open AccessIdentification and reconstitution of the origin recognition complex from Schizosaccharomyces pombe
Author(s) -
Kyeong-Yeop Moon,
Delong Kong,
Joon-Kyu Lee,
Santanu Raychaudhuri,
Jerard Hurwitz
Publication year - 1999
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.96.22.12367
Subject(s) - schizosaccharomyces pombe , biology , protein subunit , schizosaccharomyces , saccharomyces cerevisiae , dna , origin recognition complex , microbiology and biotechnology , cloning (programming) , dna replication , genetics , biochemistry , yeast , eukaryotic dna replication , gene , computer science , programming language
The origin recognition complex (ORC), first identified inSaccharomyces cerevisiae (sc), is a six-subunit protein complex that binds to DNA origins. Here, we report the identification and cloning of cDNAs encoding the six subunits of the ORC ofSchizosaccharomyces pombe (sp). Sequence analyses revealed that spOrc1, 2, and 5 subunits are highly conserved compared with their counterparts fromS. cerevisiae ,Xenopus ,Drosophila , and human. In contrast, both spOrc3 and spOrc6 subunits are poorly conserved. As reported by Chuang and Kelly [(1999)Proc. Natl. Acad. Sci. USA 96, 2656–2661], the C-terminal region of spOrc4 is also conserved whereas the N terminus uniquely contains repeats of a sequence that binds strongly to AT-rich DNA regions. Consistent with this, extraction ofS. pombe chromatin with 1 M NaCl, or after 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 reconstitutedin vitro with all six recombinant subunits expressed in the rabbit reticulocyte system. The association of spOrc4p with the other subunits required the removal of DNA from reaction mixture by DNase I. This suggests that 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 contribute to our understanding of the sequence-specific recognition required for the initiation of DNA replication inS. pombe .