The MluI cell cycle box (MCB) motifs, but not damage-responsive elements (DREs), are responsible for the transcriptional induction of the rhp51+ gene in response to DNA replication stress.

Abstract

DNA replication stress induces the transcriptional activation of rhp51 +, a fission yeast recA homolog required for repair of DNA double strand breaks. However, the mechanism by which DNA replication stress activates rhp51 + transcription is not understood. The promoter region of rhp51 + contains two damage-responsive elements (DREs) and two MluI cell cycle box (MCB) motifs. Using luciferase reporter assays, we examined the role of these elements in rhp51 + transcription. The full-length rhp51 + promoter and a promoter fragment containing MCB motifs only, but not a fragment containing DREs, mediated transcriptional activation upon DNA replication stress. Removal of the MCB motifs from the rhp51 + promoter abolished the induction of rhp51 + transcription by DNA replication stress. Consistent with a role for MCB motifs in rhp51 + transcription activation, deletion of the MBF (MCB-binding factor) co-repressors Nrm1 and Yox1 precluded rhp51 + transcriptional induction in response to DNA replication stress. Using cells deficient in checkpoint signaling molecules, we found that the Rad3-Cds1/Chk1 pathway partially mediated rhp51 + transcription in response to DNA replication stress, suggesting the involvement of unidentified checkpoint signaling pathways. Because MBF is critical for G1/S transcription, we examined how the cell cycle affected rhp51 + transcription. The transcription of rhp51 + and cdc18 +, an MBF-dependent G1/S gene, peaked simultaneously in synchronized cdc25-22 cells. Furthermore, DNA replication stress maintained transcription of rhp51 + similarly to cdc18 +. Collectively, these results suggest that MBF and its regulators mediate rhp51 + transcription in response to DNA replication stress, and underlie rhp51 + transcription at the G1/S transition.