The characterization of Dbf4 interactions and roles in genome replication and stability in Saccharomyces cerevisiae

Abstract

Dbf4-dependent kinase (DDK) is a complex composed of the Cdc7 kinase and its regulatory subunit, Dbf4. Initiation of eukaryotic DNA replication requires phosphorylation of Mcm2-7 helicase by DDK. Due to its essential role in replication, DDK is also an S phase checkpoint response target of Rad53 kinase. Dbf4 is conserved among eukaryotes and interacts with its ligands to regulate DNA replication initiation. In the studies detailed in this thesis, I characterized the molecular details of Dbf4 binding with its targets and their in vivo significance in the budding yeast Saccharomyces cerevisiae. Previous structural studies revealed that Dbf4 HBRCT directly binds to the Rad53 FHA1 domain in a non-canonical way as part of the DNA damage checkpoint response. This interaction is phosphorylation independent, which is novel and distinctive since both BRCT and FHA are known as a phospoepitope recognition module. Work included in this thesis presents the crystal structures of Rad53 FHA1 bound to Dbf4, in the presence of a phosphorylated Cdc7 peptide. FHA1 was shown to interact with both subunits of DDK by using the canonical binding mode to recognize the Cdc7 phosphopeptide and a non-canonical surface to interact with Dbf4. Two interfaces that mediate the Dbf4-Rad53 interaction were also identified and revealed that HBRCT and FHA1 do not contribute equally in this binding. To prevent precocious DDK activation in G1 phase, Rif1 targets Glc7 phosphatase to origins of DNA replication where it dephosphorylates Mcm2-7. As cells approach S phase, both DDK and cyclin dependent kinase (CDK) phosphorylate the Glc7 binding region of Rif1, inhibiting the Rif1-Glc7 association. A previous study showed that the Dbf4 N-terminus interacts with the Rif1 C-terminus to mediate DDK phosphorylation. This Dbf4 region includes a HBRCT domain that binds to Rad53 during checkpoint conditions. Two-hybrid analysis revealed the Dbf4 HBRCT domain mediates interaction with Rif1 in a fashion distinct from the way it promotes the Dbf4-Rad53 association. The Rif1 C-terminus is also known to bind the telomere-associated ligand Rap1. A combination of bioinformatics and two-hybrid analysis uncovered a proline-rich (PPDSPP) C-terminal Rif1 motif that is required for Dbf4 binding, but not Rap1. Combining a deletion of this motif (∆PPDSPP) with mutation of RIF1 CDK target sites, (5A), resulted in a slow growth phenotype and hypersensitivity to bleocin and phleomycin, suggesting impaired function in DNA double-strand break repair. rif1 5A∆PPDSPP was further combined with mutations affecting either non-homologous end-joining (NHEJ) or homologous recombination (HR) and revealed that HR disruption causes increased rif1 5A∆PPDSPP sensitivity to a range of genotoxic agents while NHEJ disruption resulted in a striking rescue of this strain. Therefore, Rif1, besides being modulated by DDK and CDK in DNA replication initiation, appears to be regulated by the same kinases during cellular recovery from genotoxic stress. Recent reports on Mrc1/Claspin suggest a novel checkpoint-independent function in DNA replication initiation in fission yeast and humans. This role involves the Mrc1/Claspin interaction with DDK through its Cdc7 subunit. As part of the investigations conducted for this thesis, two-hybrid assays were carried out and demonstrated that budding yeast Mrc1 associates with DDK via Dbf4, not Cdc7. Moreover, preliminary in vivo data revealed that deletion of budding yeast MRC1 exacerbated cell growth when DDK activity was compromised, which is different from what was previously seen in fission yeast. Therefore, our initial findings indicate that Mrc1 may function differently in budding and fission yeast DNA replication.