Yeast two-hybrid analysis of the origin recognition complex ofSaccharomyces cerevisiae: interaction between subunits and identification of binding proteins

Abstract

Origin recognition complex (ORC), a six-protein complex (Orc1p-6p), is the most likely initiator of chromosomal DNA replication in eukaryotes. Although ORC of Saccharomyces cerevisiae has been studied extensively from biochemical and genetic perspectives, its quaternary structure remains unknown. Previous studies suggested that ORC has functions other than DNA replication, such as gene silencing, but the molecular mechanisms of these functions have not been determined. In this study, we used yeast two-hybrid analysis to examine the interaction between ORC subunits and to search for ORC-binding proteins. As well as the known Orc4p-Orc5p interaction, we revealed strong interactions between Orc2p and Ord3p (2p-3p), Orc2p and Ord5p (2p-5p), Orc2p and Ord6p (2p-6p) and Orc3p and Ord6p (3p-6p) and weaker interactions between Orc1p and Ord4p (1p-4p), Orc3p and Ord4p (3p-4p), Orc2p and Ord3p (3p-5p) and Orc5p and Ord3p (5p-6p). These results suggest that 2p-3p-6p may form a core complex. Orc2p and Orc6p are phosphorylated in vivo, regulating initiation of DNA replication. However, replacing the phosphorylated amino acid residues with others that cannot be phosphorylated, or that mimic phosphorylation, did not affect subunit interactions. We also identified several proteins that interact with ORC subunits; Sir4p and Mad1p interact with Orc2p; Cac1p and Ykr077wp with Orc3p; Rrm3p and Swi6p with Orc5p; and Mih1p with Orc6p. We discuss roles of these interactions in functions of ORC.