Characterization of the Interaction Between Dbf4 and Rad53 During Replication Stress in Budding Yeast

Abstract

<p>All living things must replicate their DNA. Despite being essential for life, this process is also inherently dangerous. Replication stress, which induces replication fork stalling, is an unavoidable risk that can trigger potentially harmful changes to the genome. Eukaryotes have a replication checkpoint pathway that stabilizes stalled replication forks to prevent damage. One of the critical protein interactions in this pathway, between Dbf4 and Rad53, pauses the cell cycle in budding yeast. This is important to give the cell time to recover from stress. The molecular details of this interaction were investigated to shed light on how this association is regulated by the cell. The structure of an N-terminal domain from Dbf4 was solved through X-ray crystallography and discovered to have a modified <em>BR</em>CA-1 <em>C</em>-<em>t</em>erminal (BRCT) fold, which included an additional N-terminal helix. This domain could interact with the <em>F</em>ork<em>H</em>ead <em>A</em>ssociated 1 (FHA1) domain from Rad53 <em>in vitro</em>, and the additional helix was necessary for complex formation. Although the FHA1 domain has a well-characterized binding site for phospho-epitopes, a combination of chemical cross-linking and NMR spectroscopy experiments demonstrated that the N-terminal domain from Dbf4 is contacting an alternative surface. However, the full-length Dbf4 protein <em>in vivo</em> may be contacting both this distal site and the phospho-epitope binding pocket. This bipartite interaction between Dbf4 and Rad53 would lend specificity to the complex and also suggests a kinase may be regulating the association. As FHA and BRCT domains are prevalent in eukaryotic nuclear proteins, these findings are instructive for how these domains mediate interactions in other signaling pathways.</p>