|dc.description.abstract||Since the discovery of the Forkhead-associated (FHA) domain in 1995, research groups have pursued the characterization of FHA domains and the interactions that they regulate through the lens of their pThr-epitope binding specificity. Over the past two decades, isolated cases of FHA domains that were not just relying on the pThr-epitope binding site for the establishment of interactions have been reported. One case from the Duncker Lab reported that the interaction between the FHA1 domain of Rad53 and the H-BRCT domain of Dbf4 did not use the Rad53 FHA1 pThr-epitope binding site for the establishment of the interaction. Instead, a conserved patch of residues located on the distal lateral surface was responsible for the interaction between Rad53 and Dbf4. Since the publication of that discovery, the prevalence of such non-canonical interactions has been a persistent question. This thesis aimed to address instances of additional cases where an FHA domain was responsible for mediating a protein-protein interaction in a way that was independent of the pThr-epitope binding site. Specifically, the goals were to: (1) explore whether the entire list of budding yeast FHA domains had been collected, (2) characterize the known FHA domains for lateral surface conservation via the identification and distinguishment of candidate residues on the lateral surfaces that could be involved in protein-protein interactions as well as the pThr-epitope binding site, (3) explore whether other H-BRCT domains besides that of Dbf4 and Sir4 could be identified, in case the non-canonical nature of the Rad53-Dbf4 interaction was found to be a consequence of the H-BRCT domain structure, and (4) demonstrate instances of other yeast FHA domain-containing proteins that utilize the lateral surface(s) of the FHA domain as an interface to mediate protein-protein interaction.
Yeast two-hybrid assays looking at the interactions between Dun1 and two of its ligands Dif1 and Sml1 showed that the Dun1 FHA domain utilizes its distal lateral surface in conjunction with the pThr-epitope binding site to mediate ligand binding and efficiency. Loss of interactions with Dif1 and Sml1 in response to mutations of the pThr-epitope binding site and distal lateral surface resulted in hypersensitivity to methyl methanesulfonate (MMS), a DNA alkylating agent, and hydroxyurea (HU), which depletes dNTP pools, as well as sensitivity to DNA double strand break (DSB)-inducing agents bleomycin and phleomycin. Analysis of the sequence and structural conservation of all known budding yeast FHA domains revealed two ‘motifs’, the first being the ‘GR motif’ for the identification of the conserved arginine residue of the pThr-epitope binding site. The second motif was an ‘SxxH motif’ located downstream of the ‘GR motif’. When the conserved histidine residue was mutated to alanine, the Dun1-Dif1 interaction was significantly reduced. Comparing the FHA domains of budding yeast paralogs and a human homolog: Rad53, Dun1, Mek1, and Chk2, a different secondary motif was found that consisted of a highly conserved cysteine and proline residue. Mutation of the cysteine residue also resulted in a significant reduction in the Dun1-Dif1 interaction, although not to the same extent as mutation of the conserved histidine. Alignment of the FHA domain structures of the Rad53 FHA1, Dun1, Mek1, and Chk2 FHA domains showed positional conservation of residues that are known to be involved in FHA domain-mediated protein-protein interactions, indicating evolutionary conservation of lateral surface regions that could also be related to protein function(s). Novel FHA and H-BRCT domains were not identified, although further investigation could lead to a better understanding of the difference and evolutionary development of tandem BRCT domains and the alpha-helical linker region that can be found between tandem BRCT domains. Characterization of the meiosis specific Rad53 paralog, Mek1, revealed that the proximal lateral surface of the Mek1 FHA domain is involved in the establishment of the Mek1-Ndt80 interaction. Loss of interaction as a result of mutating residues on the β-strands of the proximal lateral surface led to compromised interhomolog repair of Spo11-induced DSBs and Mek1 kinase activity whereas mutation of residues on an α-helical loop connecting those β-strands specifically abrogated the Mek1-Ndt80 interaction without impairing Mek1 kinase activity.||en