Isolation of <i>ORC6</i> , a Component of the Yeast Origin Recognition Complex by a One-Hybrid System

Abstract

Here a method is described to identify genes encoding proteins that recognize a specific DNA sequence. A bank of random protein segments tagged with a transcriptional activation domain is screened for proteins that can activate a reporter gene containing the sequence in its promoter. This strategy was used to identify an essential protein that interacts in vivo with the yeast origin of DNA replication. Matches between its predicted amino acid sequence and peptide sequence obtained from the 50-kilodalton subunit of the yeast origin recognition complex (ORC) established that the gene isolated here, ORC6, encodes this subunit. These observations provide evidence that ORC recognizes yeast replication origins in vivo.