| Summary: | In response to DNA damage, a signaling cascade is activated within cells that promotes cell cycle arrest in order to provide adequate time for DNA repair to occur. Replication Factor A (RFA) is an essential, heterotrimeric complex that acts as a primary sensor of DNA damage, localizes to sites of and binds to broken DNA, and serves as a platform for several DNA damage repair proteins. RFA-coated ssDNA leads to activation of the sensor kinase Mec1. Activation of Mec1 leads directly to phosphorylation of the checkpoint regulator Rad53, which in turn activates several downstream effectors that halt cell growth and promote DNA repair. In response to a permanent, irreparable DNA lesion yeast cells undergo Rad53-dependent checkpoint arrest for approximately 12 hours. After this prolonged arrest, cell cycle restart occurs sometime between 12-15 hours, promoting mitosis despite the presence of an unrepaired lesion. This aberrant checkpoint exit is known as checkpoint adaptation. Checkpoint adaptation deficiency, or the inability to override the established Rad53-dependent checkpoint, can be conferred through deletion of non-essential but important genes involved in DNA damage repair (e.g., ku70, rdh54). The molecular mechanism(s) by which checkpoint adaptation occurs remains poorly understood; however, a single point mutation (K45E) in the Rfa1 subunit of the RFA complex is capable of rescuing adaptation deficiencies. During the DNA damage response, the RFA complex is known to be post-translationally modified through phosphorylation, ubiquitination, and sumoylation. Despite robust characterization of these modifications to RFA, the physiological role of phosphorylation within the N-terminal (NT) domain of the second subunit of the RFA complex, Rfa2, remains elusive. My studies using phospho-mimetic Rfa2 mutants demonstrates that phosphorylation of the NT is: 1) dispensable for activation of the Rad53-dependent checkpoint, 2) is readily detected during the temporal window within which checkpoint adaptation occurs and 3) promotes checkpoint adaptation in all adaptation-deficient mutations tested. Furthermore, analysis of Rfa2 phospho-mutants in sensor kinase deletions mec1 and tel1 reveal phosphorylation of the Rfa2 NT may directly impede Mec1 activity, thereby inhibiting maintenance of the Rad53-dependent checkpoint and providing a mechanism by which cells can escape an established DNA damage-dependent checkpoint.
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