Role of Multifunctional Autonomously Replicating Sequence Binding Factor 1 in the Initiation of DNA Replication and Transcriptional Control in Saccharomyces cerevisiae

Abstract

Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN₅ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs. Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity. To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated. Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature. At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the as-acting ABF1 binding sites analyzed previously by numerous investigators. Flow cytometric analysis and in vivo DNA labeling experiments on an α-factor synchronized abf1 (Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G₁ through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain. RNA synthesis is also reduced in the abf1 (Ts) strains. In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1 (Ts) strain at the semipermissive temperature. These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation.