A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Giuseppe Melacini,Madoka Akimoto,Bernard P. Duncker,Brendan J. McConkey,Alba Guarné,Rajeevan Selvaratnam,Lindsay A. Matthews,Darryl R. Jones

doi:10.1074/jbc.m113.517060

Giuseppe Melacini, Madoka Akimoto + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m113.517060

Copy DOI

Abstract

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily 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 that is critical to suppress late origin firing and to 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.

Highlights

The interaction between the checkpoint kinase Rad53 and Dbf4 is critical to suppress late origin firing and to stabilize stalled forks during replication stress
We found that the FHA1 domain of Rad53 can bind simultaneously to the HBRCT domain of Dbf4 and a pThr-containing phosphoepitope, suggesting that a bipartite interaction may modulate the interaction between Rad53 and Dbf4 in vivo
We found that the presence of phosphorylated Cdc7 peptide (pPEP) caused chemical shift perturbations consistent with phosphopeptide binding, whereas the non-phosphorylated version of the peptide (PEP) did not induce any changes in the 15NFHA1 Heteronuclear single quantum correlation (HSQC) spectrum (Fig. 7)

Summary

Background

The interaction between the checkpoint kinase Rad and Dbf is critical to suppress late origin firing and to stabilize stalled forks during replication stress. Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins They function primarily as phosphoepitope recognition modules but can mediate non-canonical interactions. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. We found that the FHA1 domain of Rad can bind simultaneously to the HBRCT domain of Dbf and a pThr-containing phosphoepitope, suggesting that a bipartite interaction may modulate the interaction between Rad and Dbf in vivo This dual binding mechanism and its functional implications for the replication checkpoint are discussed

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION