Differential Regulation of JAMM Domain Deubiquitinating Enzyme Activity within the RAP80 Complex

BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.     

The Lys63-specific Deubiquitinating Enzyme BRCC36 Is Regulated by Two Scaffold Proteins Localizing in Different Subcellular Compartments

BRCC36 is a member of the JAMM/MPN⁺ family of zinc metalloproteases that specifically cleaves Lys 63-linked polyubiquitin chains in vitro. We and others showed previously that BRCC36 is a component of the BRCA1-A complex, which consists of RAP80, CCDC98/ABRAXAS, BRCC45/BRE, MERIT40/NBA1, BRCC36, and BRCA1. This complex participates in the regulation of BRCA1 localization in response to DNA damage. Here we provide evidence indicating that BRCC36 regulates the abundance of Lys⁶³-linked ubiquitin chains at chromatin and that one of its substrates is diubiquitinated histone H2A. Moreover, besides interacting with CCDC98 within the BRCA1-A complex, BRCC36 also associates with another protein KIAA0157, which shares significant sequence homology with CCDC98. Interestingly, although CCDC98 functions as an adaptor of BRCC36 and regulates BRCC36 activity in the nucleus, KIAA0157 mainly localizes in cytosol and activates BRCC36 in the cytoplasm. Moreover, these two complexes appear to exist in fine balance in vivo because reduction of KIAA0157 expression led to an increase of the BRCA1-A complex in the nucleus. Together, these results suggest that scaffold proteins not only participate in the regulation of BRCC36 activity but also determine its subcellular localization and cellular functions.     

The in vivo dynamic organization of BRCA1-A complex proteins at DNA damage-induced nuclear foci

The breast cancer associated gene 1 (BRCA1)‐A protein complex assembles at DNA damage‐induced nuclear foci to facilitate repair of double‐stranded breaks. Here, we describe the first systematic comparison of the dynamics, copy number and organization of its core components at foci. We show that the protein pools at individual foci generally comprise a small immobile fraction (～20%) and larger mobile fraction (～80%), which together occupy the same focal space but exist at different densities. In the mobile fraction, Abraxas (CCDC98) and the heterodimer BARD1–BRCA1 share similar rates of dynamic exchange (complete turnover in ～500 seconds). In contrast, RAP80, which is required for initial foci assembly, was more dynamic with 25‐fold faster turnover at mature foci. In addition, Abraxas, BARD1, BRCA1 and Merit40 (NBA1) were stably retained in the immobile fraction of foci under conditions causing loss of BRCC36 and RAP80, suggesting a shift to RAP80‐independent localization after foci formation. These results, combined with our finding that RAP80 (～1200 copies per focus) is twofold more abundant than Abraxas/BARD1/BRCA1 at foci, suggest new models defining the dynamic organization of BRCA1‐A complex at mature foci, wherein the unusually fast turnover of RAP80 may contribute to its regulation of BRCA1‐dependent DNA repair.     

Regulation of BRCC, a holoenzyme complex containing BRCA1 and BRCA2, by a signalosome-like subunit and its role in DNA repair

We have isolated a holoenzyme complex termed BRCC containing BRCA1, BRCA2, and RAD51. BRCC not only displays increased association with p53 following DNA damage but also ubiquitinates p53 in vitro. BRCC36 and BRCC45 are novel components of the complex with sequence homology to a subunit of the signalosome and proteasome complexes. Reconstitution of a recombinant four-subunit complex containing BRCA1/BARD1/BRCC45/BRCC36 revealed an enhanced E3 ligase activity compared to that of BRCA1/BARD1 heterodimer. In vivo, depletion of BRCC36 and BRCC45 by the small interfering RNAs (siRNAs) resulted in increased sensitivity to ionizing radiation and defects in G2/M checkpoint. BRCC36 shows aberrant expression in sporadic breast tumors. These findings identify BRCC as a ubiquitin E3 ligase complex that enhances cellular survival following DNA damage.     

Loss of BRCA1-A complex function in RAP80 null tumor cells

Receptor Associated Protein 80 (RAP80) is a subunit of the BRCA1-A complex and targets BRCA1 to DNA damage sites in response to DNA double strand breaks. Since mutations of BRCA1 are associated with familial ovarian cancers, we screened 26 ovarian cancer-derived cell lines for RAP80 mutations and found that TOV-21G cells harbor a RAP80 mutation (c.1107G >A). This mutation generates a stop codon at Trp369, which deletes the partial AIR region and the C-terminal zinc fingers of RAP80. Interestingly, both the mutant and wild type alleles of RAP80 lose their expression due to promoter hypermethylation, suggesting that TOV-21G is a RAP80-null cell line. In these cells, not only is the BRCA1-A complex disrupted, but the relocation of the remaining subunits in the BRCA1-A complex including BRCA1, CCDC98, NBA1, BRCC36 and BRE is significantly suppressed. Moreover, TOV-21G cells are hypersensitive to ionizing radiation, which is due to the compromised DNA damage repair capacity in these cells. Reconstitution of TOV-21G cells with wild type RAP80 rescues these cellular defects in response to DNA damage. Thus, our results demonstrate that RAP80 is a scaffold protein in the BRCA1-A complex. Identification of TOV-21G as a RAP80 null tumor cell line will be very useful for the study of the molecular mechanism in DNA damage response.     

The BRCA1-RAP80 complex regulates DNA repair mechanism utilization by restricting end resection

The tumor suppressor protein BRCA1 is a constituent of several different protein complexes and is required for homology-directed repair (HDR) of DNA double strand breaks (DSBs). The most recently discovered BRCA1-RAP80 complex is recruited to ubiquitin structures on chromatin surrounding the break. Deficiency of any member of this complex confers hypersensitivity to DNA-damaging agents by undefined mechanisms. In striking contrast to other BRCA1-containing complexes that are known to promote HDR, we demonstrate that the BRCA1-RAP80 complex restricts end resection in S/G(2) phase of the cell cycle, thereby limiting HDR. RAP80 or BRCC36 deficiency resulted in elevated Mre11-CtIP-dependent 5' end resection with a concomitant increase in HDR mechanisms that rely on 3' single-stranded overhangs. We propose a model in which the BRCA1-RAP80 complex limits nuclease accessibility to DSBs, thus preventing excessive end resection and potentially deleterious homology-directed DSB repair mechanisms that can impair genome integrity.     

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes.     

BRCA1 tumor suppressor network: focusing on its tail

ABSTRACT: Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.     

Interactions between human BRCA2 protein and the meiosis-specific recombinase DMC1

Germline mutations in BRCA2 predispose to hereditary breast cancers. BRCA2 protein regulates recombinational repair by interaction with RAD51 via a series of degenerate BRC repeat motifs encoded by exon 11 (BRCA2(996-2113)), and an unrelated C-terminal domain (BRCA2(3265-3330)). BRCA2 is also required for meiotic recombination. Here, we show that human BRCA2 binds the meiosis-specific recombinase DMC1 and define the primary DMC1 interaction site to a 26 amino-acid region (BRCA2(2386-2411)). This region is highly conserved in BRCA2 proteins from a variety of mammalian species, but is absent in BRCA2 from Arabidopsis thaliana, Caenorhabditis elegans, and other eukaryotes. We demonstrate the critical importance of Phe2406, Pro2408, and Pro2409 at the conserved motif (2404)KVFVPPFK(2411). This interaction domain, defined as the PhePP motif, promotes specific interactions between BRCA2 and DMC1, but not with RAD51. Thus, the RAD51 and DMC1 interaction domains on BRCA2 are distinct from each other, allowing coordinated interactions of the two recombinases with BRCA2 at meiosis. These results lead us to suggest that BRCA2 is a universal regulator of RAD51/DMC1 recombinase actions.     

CtIP protein dimerization is critical for its recruitment to chromosomal DNA double-stranded breaks

CtIP (CtBP-interacting protein) associates with BRCA1 and the Mre11-Rad50-Nbs1 (MRN) complex and plays an essential role in homologous recombination (HR)-mediated DNA double-stranded break (DSB) repair. It has been described that CtIP forms dimers in mammalian cells, but the biological significance is not clear. In this study, we identified a conserved motif in the N terminus of CtIP, which is required for dimer formation. We further showed that CtIP mutants impaired in forming dimers are strongly defective in HR, end resection, and activation of the ataxia telangiectasia and Rad3-related pathway, without notable change of CtIP interactions with BRCA1 or Nbs1. In addition to HR, CtIP dimerization is also required for microhomology-mediated end joining. Live cell imaging of enhanced GFP-tagged CtIP demonstrates that the CtIP dimerization mutant fails to be localized to DSBs, whereas placing a heterologous dimerization motif to the dimerization mutant restores CtIP recruitment to DSBs. These studies suggest that CtIP dimer formation is essential for its recruitment to DSBs on chromatin upon DNA damage. Furthermore, DNA damage-induced phosphorylation of CtIP is significantly reduced in the CtIP dimerization mutants. Therefore, in addition to the C-terminal conserved domains critical for CtIP function, the dimerization motif on the N terminus of CtIP is also conserved and essential for its function in DNA damage responses. The severe repair defects of CtIP dimerization mutants are likely due to the failure in localization to chromosomal DSBs upon DNA damage.     

BRE over-expression promotes growth of hepatocellular carcinoma

BRE, also known as TNFRSF1A modulator and BRCC45, is an evolutionarily highly conserved protein. It is a death receptor-associated protein in cytoplasm and a component of BRCA1/2-containing DNA repair complex in nucleus. BRE was found to have anti-apoptotic activity. Over-expression of BRE by transfection promoted survival of cell lines against apoptotic induction; whereas depletion of the protein by siRNA resulted in the opposite. In vivo anti-apoptotic activity of BRE was demonstrated by significant attenuation of Fas-induced acute fulminant hepatitis in transgenic mice expressing the human protein specifically in the liver. BRE was also implicated in tumor promotion by the accelerated tumor growth of Lewis Lung carcinoma transfected with human BRE; and by high expression of BRE specifically in the tumoral regions of human hepatocellular carcinoma (HCC). The present study was to test directly if transgenic expression of BRE in livers could promote HCC development in neonatal diethylnitrosamine model. By 8 months after tumor induction, the maximal sizes of tumor nodules of transgenic mice were significantly larger than those of the non-transgenic controls, although the numbers of tumor nodules between the two groups did not significantly differ. Importantly, as in human HCC, the mouse endogenous BRE level was up-regulated in mouse HCC nodules. These results show that BRE over-expression can indeed promote growth, though not initiation, of liver tumors. Furthermore, the common occurrence of BRE over-expression in human and mouse HCC suggests that up-regulation of BRE is functionally important in liver tumor development.     

A Dual Interaction between the DNA Damage Response Protein MDC1 and the RAG1 Subunit of the V(D)J Recombinase

The first step in V(D)J recombination is the formation of specific DNA double-strand breaks (DSBs) by the RAG1 and RAG2 proteins, which form the RAG recombinase. DSBs activate a complex network of proteins termed the DNA damage response (DDR). A key early event in the DDR is the phosphorylation of histone H2AX around DSBs, which forms a binding site for the tandem BRCA1 C-terminal (tBRCT) domain of MDC1. This event is required for subsequent signal amplification and recruitment of additional DDR proteins to the break site. RAG1 bears a histone H2AX-like motif at its C terminus (R1Ct), making it a putative MDC1-binding protein. In this work we show that the tBRCT domain of MDC1 binds the R1Ct motif of RAG1. Surprisingly, we also observed a second binding interface between the two proteins that involves the Proline-Serine-Threonine rich (PST) repeats of MDC1 and the N-terminal non-core region of RAG1 (R1Nt). The repeats-R1Nt interaction is constitutive, whereas the tBRCT-R1Ct interaction likely requires phosphorylation of the R1Ct motif of RAG1. As the C terminus of RAG1 has been implicated in inhibition of RAG activity, we propose a model in which phosphorylation of the R1Ct motif of RAG1 functions as a self-initiated regulatory signal.     

Rap80 protein recruitment to DNA double-strand breaks requires binding to both small ubiquitin-like modifier (SUMO) and ubiquitin conjugates

Ubiquitin (Ub) modifications at sites of DNA double-strand breaks (DSBs) play critical roles in the assembly of signaling and repair proteins. The Ub-interacting motif (UIM) domain of Rap80, which is a component of the BRCA1-A complex, interacts with Ub Lys-63 linkage conjugates and mediates the recruitment of BRCA1 to DSBs. Small ubiquitin-like modifier (SUMO) conjugation also occurs at DSBs and promotes Ub-dependent recruitment of BRCA1, but its molecular basis is not clear. In this study, we identified that Rap80 possesses a SUMO-interacting motif (SIM), capable of binding specifically to SUMO2/3 conjugates, and forms a tandem SIM-UIM-UIM motif at its N terminus. The SIM-UIM-UIM motif binds to both Ub Lys-63 linkage and SUMO2 conjugates. Both the SIM and UIM domains are required for efficient recruitment of Rap80 to DSBs immediately after damage and confer cellular resistance to ionizing radiation. These findings propose a model in which SUMO and Ub modification is coordinated to recruit Rap80 and BRCA1 to DNA damage sites.     

Cell cycle‐regulated ubiquitination of tankyrase 1 by RNF8 and ABRO1/BRCC36 controls the timing of sister telomere resolution

Timely resolution of sister chromatid cohesion in G2/M is essential for genome integrity. Resolution at telomeres requires the poly(ADP‐ribose) polymerase tankyrase 1, but the mechanism that times its action is unknown. Here, we show that tankyrase 1 activity at telomeres is controlled by a ubiquitination/deubiquitination cycle depending on opposing ubiquitin ligase and deubiquitinase activities. In late S/G2 phase, the DNA damage‐responsive E3 ligase RNF8 conjugates K63‐linked ubiquitin chains to tankyrase 1, while in G1 phase such ubiquitin chains are removed by BRISC, an ABRO1/BRCC36‐containing deubiquitinase complex. We show that K63‐linked ubiquitin chains accumulate on tankyrase 1 in late S/G2 to promote its stabilization, association with telomeres, and resolution of cohesion. Timing of this posttranslational modification coincides with the ATM‐mediated DNA damage response that occurs on functional telomeres following replication in G2. Removal of ubiquitin chains is controlled by ABRO1/BRCC36 and occurs as cells exit mitosis and enter G1, ensuring that telomere cohesion is not resolved prematurely in S phase. Our studies suggest that a cell cycle‐regulated posttranslational mechanism couples resolution of telomere cohesion with completion of telomere replication to ensure genome integrity.     

Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control

The diverse roles of TopBP1 in DNA replication and checkpoint signaling are associated with the scaffolding ability of TopBP1 to initiate various protein-protein interactions. The recognition of the BACH1/FANCJ helicase by TopBP1 is critical for the activation of the DNA replication checkpoint at stalled replication forks and is facilitated by the C-terminal tandem BRCT7/8 domains of TopBP1 and a phosphorylated Thr(1133) binding motif in BACH1. Here we provide the structural basis for this interaction through analysis of the x-ray crystal structures of TopBP1 BRCT7/8 both free and in complex with a BACH1 phospho-peptide. In contrast to canonical BRCT-phospho-peptide recognition, TopBP1 BRCT7/8 undergoes a dramatic conformational change upon BACH1 binding such that the two BRCT repeats pivot about the central BRCT-BRCT interface to provide an extensive and deep peptide-binding cleft. Additionally, we provide the first structural mechanism for Thr(P) recognition among BRCT domains. Together with systematic mutagenesis studies, we highlight the role of key contacts in governing the unique specificity of the TopBP1-BACH1 interaction.     

Chromodomain helicase DNA-binding protein 4 (CHD4) regulates homologous recombination DNA repair, and its deficiency sensitizes cells to poly(ADP-ribose) polymerase (PARP) inhibitor treatment

To ensure genome stability, cells have evolved a robust defense mechanism to detect, signal, and repair damaged DNA that is generated by exogenous stressors such as ionizing radiation, endogenous stressors such as free radicals, or normal physiological processes such as DNA replication. Homologous recombination (HR) repair is a critical pathway of repairing DNA double strand breaks, and it plays an essential role in maintaining genomic integrity. Previous studies have shown that BRIT1, also known as MCPH1, is a key regulator of HR repair. Here, we report that chromodomain helicase DNA-binding protein 4 (CHD4) is a novel BRIT1 binding partner that regulates the HR repair process. The BRCA1 C-terminal domains of BRIT1 are required for its interaction with CHD4. Depletion of CHD4 and overexpression of the ATPase-dead form of CHD4 impairs the recruitment of BRIT1 to the DNA damage lesions. As a functional consequence, CHD4 deficiency sensitizes cells to double strand break-inducing agents, reduces the recruitment of HR repair factor BRCA1, and impairs HR repair efficiency. We further demonstrate that CHD4-depleted cells are more sensitive to poly(ADP-ribose) polymerase inhibitor treatment. In response to DNA damage induced by poly(ADP-ribose) polymerase inhibitors, CHD4 deficiency impairs the recruitment of DNA repair proteins BRIT1, BRCA1, and replication protein A at early steps of HR repair. Taken together, our findings identify an important role of CHD4 in controlling HR repair to maintain genome stability and establish the potential therapeutic implications of targeting CHD4 deficiency in tumors.     

A PP1-binding motif present in BRCA1 plays a role in its DNA repair function

Protein phosphatase 1alpha (PP1alpha) regulates phosphorylation of BRCA1, which contains a PP1-binding motif (898)KVTF(901). Mutation of this motif greatly reduces the interaction between BRCA1 and PP1alpha. Here we show that mutation of the PP1-binding motif abolishes the ability of BRCA1 to enhance survival of Brca1-deficient mouse mammary tumor cells after DNA damage. The Rad51 focus formation and comet assays revealed that the DNA repair function of BRCA1 was impaired when the PP1-binding motif was mutated. Analysis of subnuclear localization of GFP-tagged BRCA1 demonstrated that mutation of the PP1-binding motif affected BRCA1 redistribution in response to DNA damage. BRCA1 is required for the formation of Rad51 subnuclear foci after DNA damage. Mutation of the PP1-binding motif in BRCA1 also affected recruitment of Rad51 to sites of DNA damage. Consistent with these findings, knockdown of PP1alpha in BRCA1-proficient cells by small interfering RNA also significantly reduced Rad51 focus formation induced by DNA damage. Further analysis indicated that mutation of the PP1-binding motif compromised BRCA1 activities in homologous recombination. Altogether, our data implicate that interaction with PP1alpha is important for BRCA1 function in DNA repair.     

Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by tandem UIMs of RAP80

RAP80 has a key role in the recruitment of the Abraxas–BRCC36–BRCA1–BARD1 complex to DNA‐damage foci for DNA repair through specific recognition of Lys 63‐linked polyubiquitinated proteins by its tandem ubiquitin‐interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80‐UIM1‐UIM2) in complex with Lys 63‐linked di‐ubiquitin at 2.2 Å resolution. The two UIMs, UIM1 and UIM2, and the α‐helical inter‐UIM region together form a continuous 60 Å‐long α‐helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44‐centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63‐linked isopeptide bond. Our structure suggests that the inter‐UIM region forms a 12 Å‐long α‐helix that ensures that the UIMs are arranged to enable specific binding of Lys 63‐linked di‐ubiquitin. This was confirmed by pull‐down analyses using RAP80‐UIM1‐UIM2 mutants of various length inter‐UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter‐UIM region similar to that of RAP80‐UIM1‐UIM2, also selectively binds Lys 63‐linked di‐ubiquitin.     

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

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.