Checkpoint Regulation of S-Phase Transcription: A Dissertation

Abstract

The DNA replication checkpoint transcriptionally up-regulates genes that allow cells to adapt to and survive replication stress. Our results show that, in the fission yeast Schizosaccharomyces pombe, the replication checkpoint regulates the entire G1/S transcriptional program by directly regulating MBF (aka DSC1), the G1/S transcription factor. Instead of initiating a checkpoint-specific transcriptional program, the replication checkpoint targets MBF to maintain the normal G1/S transcriptional program during replication stress. We propose a mechanism for this regulation, based on in vitrophosphorylation of the Cdc10 subunit of MBF by the Cds1 replication-checkpoint kinase. Substitution of two potential phosphorylation sites with phospho-mimetic amino acids suffice to promote the checkpoint transcriptional program, suggesting that Cds1 phosphorylation directly regulates MBF-dependent transcription. The conservation of MBF between fission and budding yeast, and recent results implicating MBF as a target of the budding yeast replication checkpoint, suggest that checkpoint regulation of the MBF transcription factor may be a conserved strategy for coping with replication stress. Furthermore, the structural and regulatory similarity between MBF and E2F, the metazoan G1/S transcription factor, suggests that this checkpoint mechanism may be broadly conserved among eukaryotes. Our result shows that both the replication checkpoint and the S-phase DNA damage checkpoint are involved in activating MBF regulated S-phase gene transcription and that this coordinated transcriptional response is beneficial for survival during replication stress. I demonstrate that the beneficial role of the transcriptional response during checkpoint activation is mediated by three major MBF transcripts: cdc22, mrc1 and mik1. Mrc1 dependent stabilization of stalled fork is important during S phase arrest. However, cells ability to prevent mitosis (Mik1 dependent) along with stable fork (Mrc1 dependent) both are crucial for survival. Our data also suggest that the level of Cdc22 is a determining factor for replication checkpoint activation and when over-expressed can alleviate the effects not only in HU but also in MMS.