Probing the biological functions and interactions of the Fkh1 FHA domain in Saccharomyces cerevisiae.

摘要

The Saccharomyces cerevisiae Fkh1 protein is a multifunctional protein with roles in cell-cycle regulated transcription, DNA replication timing, as well as a role in recombination donor preference during mating-type switching. While Fkh1 has been implicated in many processes, very little is known about the mechanisms or interactions that govern its functions. The phosphothreonine-binding FHA domain of Fkh1 has been shown to be sufficient for regulating donor preference during mating-type switching. A model posits that Fkh1 mediates a long-range chromosomal interaction to promote recombination between two distant loci and that this requires an interaction between the FHA domain and a phosphorylated partner protein(s), but to date no relevant partner has been described. I have shown that the FHA domain is required for Fkh1's interaction with multiple partner proteins and is important for many different Fkh1 functions. In my studies I have found that Fkh1's interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. I have identified two threonines within Mph1 that were particularly important for this interaction. Yeast-2-hybrid analysis and in vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA ) specifically abolished the Fkh1-Mph1 interaction and altered donor preference during mating-type switching without affecting other functions of Mph1 in genome stability. Deletion of a second gene encoding a Fkh1-interacting protein, FDO1, also resulted in a change in Fkh1-dependent donor preference that was additive with deletion or mutation of MPH1 . However, deletion of both genes did not result in a change in donor preference as drastic as that resulting from mutation of FKH1, suggesting Fkh1 must interact with additional proteins to accomplish this role. I also found that the FHA domain was important for Fkh1's role in cell-cycle regulation, but no single interaction partner could account for this role. I propose that Fkh1 must interact with multiple different proteins to accomplish its role in donor preference as well as other roles in which Fkh1 has been implicated.