UCSF

When DNA is damaged, or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore, we constructed an in vivo replication-checkpoint mimic, which recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the natural replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires co-localization of Mrc1-LacI and Ddc2-LacI, and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the natural replication checkpoint, but that Ddc1 is absolutely required in the absence of Mrc1. We propose that co-localization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.