Saccharomyces cerevisiae Dbf4 Has Unique Fold Necessary for Interaction with Rad53 Kinase

Abstract

Background: Dbf4, the regulatory subunit of Cdc7, has essential roles in initiation of DNA replication and checkpoint activation. Results:We have identified the domain of Dbf4 that is necessary and sufficient for the interaction with Rad53. Conclusion:The structural integrity of this domain is essential for Dbf4-Rad53 interaction. Significance: This work suggests how single BRCT domains can be adapted to form complex functional units.

Extending the Interaction Repertoire of FHA and BRCT Domains

“…There is mounting evidence indicating that both single and tandem BRCT domains may require additional secondary structural elements to form functional units [10,12,81,82]. Some of these structural elements are required for structural stability, such as the additional C-terminal α-helix in the PARP-1 BRCT [81], while others enhance function ( Figure 7).…”

“…It is conceivable that these additional structural elements play the dual role of stabilizing the BRCT fold and enhancing its function. In fact, this is the case of S. cerevisiae Dbf4, the regulatory subunit of the Cdc7 kinase, where an α -helix immediately precedes the β1 strand and its presence is important for the stability of the domain, as well as the interaction with Rad53 during the checkpoint response [10,83]. …”

“…Another member of the kinesin-3 family, KIF1A, has a canonical phospho-epitope binding site and, yet, is also suspected of using this pocket for a phosphorylation-independent interaction [55]. Similarly, the FHA domain of S. cerevisiae Rad53 has a canonical phosphoepitope binding site, but is presumed to interact with the BRCT domain of Dbf4 in a phosphorylation-independent manner, though the molecular determinants of this interaction are unclear [10].…”

“…The ability of Dbf4 to crossover into the stress response pathway is partly due to a conserved motif at the N-terminus of the protein (motif N). Based on sequence alignments, it was a matter of debate whether motif N was a bona fide BRCT domain [9,87], however concerns were laid to rest when the crystal structure of the N-terminal region of S. cerevisiae Dbf4 was determined [10]. The structure revealed that this region of Dbf4 folds as a modified BRCT domain that requires an additional N-terminal α-helix to form a stable unit (Figure 7).…”

“…For instance, the regulatory subunit of the Cdc7 kinase, Dbf4, contains a single BRCT domain [9,10] and Dpb11, whose function is essential to activate pre-replication complexes, contains two BRCT pairs [8]. The Rfc1 subunit of the RFC complex, which functions in loading the sliding clamp onto ssDNA/dsDNA junctions during replication [11], also contains a BRCT domain required for DNA binding [12].…”