Characterization of the Rad53:Dbf4 interface 1 A NOVEL, NON-CANONICAL FHA BINDING INTERFACE MEDIATES THE INTERACTION BETWEEN Rad53 AND Dbf4* Lindsay A. Matthews 1 , Rajeevan Selvaratnam 2,4 , Darryl R. Jones 3,4 , Madoka Akimoto 2 , Brendan J. McConkey 3 , Giuseppe Melacini 1,2 , Bernard P. Duncker 3 and Alba Guarné 1,* 1 From the Department of Biochemistry and Biomedical Sciences and 2 Department of Chemistry and Chemical Biology McMaster University, Hamilton, Ontario L8S 4K1 3 From the Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3H1, Canada. 4 These two authors contributed equally to the work *Running Title: Characterization of the Rad53:Dbf4 interface To whom correspondence should be addressed: Alba Guarné, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4K1, Canada, Tel: (905) 525-9140 ext. 26394; FAX (905) 522-9033; e-mail: guarnea@mcmaster.ca Keywords: DNA replication; DNA damage response; checkpoint control; protein-protein interactions; protein domains; NMR; yeast genetics; bioinformatics. Background: The interaction between the checkpoint kinase Rad53 and Dbf4 is critical to suppress late origin firing and stabilize stalled forks during replication stress. Results: The FHA1 domain of Rad53 interacts with the BRCT domain of Dbf4 through a novel, non-canonical interface. Conclusion: FHA domains gain specificity by engaging multiple binding interfaces. Significance: Understanding how FHA domains interact with their binding partners is key to elucidate how they relay information along signaling pathways. ABSTRACT ForkHead Associated (FHA) and BRCA-1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They primarily function as phosphoepitope- recognition modules, but can also mediate non- canonical interactions. The latter are rare and only a few have been studied at a molecular level. We have identified a crucial, non- canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase, which is critical to suppress late origin firing and stabilize stalled forks during replication stress. The Rad53:Dbf4 interaction is phosphorylation-independent and involves a novel, non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope targeting modules. ForkHead Associated (FHA) domains are ubiquitous phosphoepitope-binding modules present in a number of proteins that play critical roles in the DNA damage and replication stress response (1). The replication checkpoint preserves the genomic integrity of the cell by stabilizing stalled forks, boosting DNA repair enzyme levels and pausing the cell cycle (2). Rad53 (the budding yeast homolog of the tumor suppressor Chk2) is an effector kinase with integral roles in the replication checkpoint (3). In contrast to other FHA-containing proteins, Rad53 has two FHA domains (FHA1 and FHA2) that mediate independent interactions of Rad53 with upstream and downstream branches of the checkpoint. One of the binding partners of FHA1 is Dbf4 (4), the http://www.jbc.org/cgi/doi/10.1074/jbc.M113.517060 The latest version is at JBC Papers in Press. Published on November 27, 2013 as Manuscript M113.517060 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on September 12, 2016 http://www.jbc.org/ Downloaded from by guest on September 12, 2016 http://www.jbc.org/ Downloaded from by guest on September 12, 2016 http://www.jbc.org/ Downloaded from by guest on September 12, 2016 http://www.jbc.org/ Downloaded from