Vol. 9, Issue 6, 1577-1588, June 1998

Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2-Cdc10, Controlling the Cell Cycle "Start"

Sayaka Tahara,^* Koichi Tanaka, Yasuhito Yuasa,^* and Hiroto Okayama

 ^*Department of Hygiene and Oncology, Tokyo Medical and Dental University, School of Medicine, Bunkyo-ku, Tokyo 113, Japan; and  Department of Biochemistry and Molecular Biology, The University of Tokyo Graduate School of Medicine, Bunkyo-ku, Tokyo 113, Japan

In the fission yeast Schizosaccharomyces pombe, passage from G₁ to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2⁺ gene, isolated as a multicopy suppressor of a temperature-sensitive cdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2-Cdc10 complex. To identify the rep2⁺ function and molecularly define its domain organization, we reconstituted the Res2-Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2-Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2 deletion mutant of fission yeast.