Identification of EXO1 interacting proteins
Exponentially growing HEK-293T cells were either left untreated or treated for 16 h with 2 mM hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase (RNR) causing depletion of the pool of dNTPs and early S-phase arrest. Whole cell extracts (WCE, 20 mg) were immunoprecipitated with a rabbit polyclonal antibody to EXO1 and resolved on an 8% SDS-polyacrylamide gel (Fig. 1A). Upon silver staining, the major bands were excised from the gel, submitted to proteolytic digestion and analyzed on a 4000 Q-Trap mass spectrometer. The data show that, in addition to EXO1, unique peptides for established (MSH2) and novel partners could be specifically identified (Fig. 1A and Table S1). Genuine novel EXO1 interactors consisted of the GTPase-activating protein TBC1D4, the ATP-dependent helicase RENT1(an essential factor of the nonsense-mediated decay (NMD) pathway deputed to the degradation of mRNAs containing premature stop codons), the leucine zipper protein 1 LUZP1, KAP1, a co-repressor of transcription and SUMO E3-ligase with established roles in the DNA damage response, the AMP deaminase AMPD2, and the polyadenylate-binding protein 1 PABP1 that regulates translational initiation. Interacting proteins commonly identified in mass spectrometry studies, such as NCL and HNRNPM, and listed at the Contaminant Repository for Affinity Purification site (www.crapome.org) were not considered further.
Analysis of the EXO1-KAP1 interaction
To validate the findings of mass spectrometry studies using independent methods, we focused on KAP1. Ectopic expression of OMNI-EXO1 and FLAG-KAP1 in HEK293T cells followed by immunoprecipitation with an anti-FLAG monoclonal antibody confirmed the interaction between the two proteins (Fig. 1B). Immunoprecipitation of the low abundance EXO1 protein from untreated cells or cells undergoing replication stress upon treatment with hydroxyurea (HU), revealed constitutive interaction with ectopically expressed FLAG-KAP1 (Fig. 1C). To assess whether the observed protein-protein interaction is direct or is mediated by unknown bridging proteins, we performed Far-Western blot analysis. To this end, purified recombinant EXO1 or the MutSα complex (MSH2/MSH6)- used as control were resolved by SDS-PAGE and transferred to PVDF. Upon visualization of the proteins by Ponceau-red (Fig. 1D, upper panel), the membrane was overlaid with purified recombinant FLAG-KAP1 and subsequently probed with a monoclonal antibody to the FLAG or control antibodies to EXO1, MSH6 and MSH2. The data showed a specific signal for the FLAG in the EXO1 lane only (Fig. 1D, lower panels), confirming that the observed interaction between EXO1 and KAP1 is direct. Since KAP1 was reported to bind MDM2 and contribute to the functional regulation of p53, we decided to examine whether MDM2 is also part of the EXO1-KAP1 complex. To this end, we immunoprecipitated OMNI-EXO1 from extracts of transiently transfected HEK-293T cells. The data showed that endogenous MDM2 could be found as a constitutive partner of ectopically expressed EXO1 (Fig. 1E). Immunoprecipitation of endogenous EXO1 confirmed interaction with endogenous MDM2 (Fig. 1F), strengthening the validity of this observation. The RING-domain MDM2 ubiquitin E3-ligase interacts with MDMX, which contains a non-functional RING-domain. To examine whether MDMX was also part of the protein complex, we ectopically expressed OMNI-EXO1 and HA-MDMX in HEK293T cells. Immunoprecipitation of OMNI-EXO1 from cells treated in the presence or the absence of HU showed constitutive interaction with MDMX (Fig. 1G). More importantly, we confirmed that endogenous EXO1 was able to interact with HA-MDMX (Fig. 1H). Finally, we performed Far-Western blot analysis with purified, recombinant proteins to explore molecular interactions. The data showed that MDM2 directly interacted with KAP1 but not with EXO1 (Fig. 1I, top panels, lanes 2 and 3) and that the MDM2-EXO1 interaction was only detectable upon bridging by KAP1 (Fig. 1I, bottom panels, lane 1). Co-immunoprecipitation experiments confirmed that in cells where KAP1 expression was lowered by RNA interference, interaction between EXO1 and MDM2 proteins was decreased (Fig. 1J).
To address the functional role of the EXO1-KAP1-MDM2 interaction, we attenuated expression of either KAP1 or MDM2 with specific shRNAs. The data showed that KAP1 depletion did not rescue EXO1 (Fig. 1K, top right panel, lane 2 vs. 4), the protein level of which is controlled by ubiquitylation-mediated degradation in response to HU. Similar results were obtained upon MDM2 depletion (Fig. 1K, bottom right panel, lane 2 vs. 4), suggesting that neither KAP1 nor MDM2 is involved in the control of EXO1 protein level.
Dysfunction of the machinery that signals DNA damage and/or addresses DNA repair is associated with cancer development and resistance to therapy, providing a direct demonstration of the link between genome instability and cancer. Intense effort is currently being devoted to the identification of protein complexes addressing recognition and repair of various forms of DNA damage, as well as to the elucidation of pathways transducing signals to the cell cycle machinery. This knowledge, in turn, is expected to help the development of more efficient drugs that addresses the lack of specificity and side-effects of current chemotherapeutics. The study presented here, focused on an essential component of error-free DNA repair pathways, contributes to filling this gap through the identification of novel proteins interacting with EXO1, hence expanding our current knowledge. Our data show that EXO1 is part of a multi-protein complex comprising the co-repressor of transcription and E3 SUMO ligase KAP1 and the ubiquitin E3-ligase MDM2/MDMX (Fig. 1A-1J and 1L). Interestingly, KAP1 was reported to cooperate with MDM2 in promoting p53 inactivation. However, functional studies that we conducted in cells depleted for KAP1 or MDM2 expression by RNA interference demonstrated that none of them contributes to modulating EXO1 protein level in response to stalled DNA replication (Fig. 1K). Hence, the fact that EXO1 is degraded in an ubiquitin-dependent manner upon stalled DNA replication, but that KAP1 or MDM2 have no role in this process, indicates that other ubiquitin E3-ligases control EXO1 protein level and, in turn, the extent of DNA resection at sites of damage. A recent study reporting the ability of SCF-Cyclin F to control EXO1 stability upon UV-damage, but not in response to ionizing radiation, indicates that distinct ubiquitin-dependent pathways may be operative in different settings.
The low abundance of EXO1, both in yeast (estimated to ~800 molecules per cell in yeast, http://www.yeastgenome.org/) and in humans (www.proteinatlas.org/), compared to the high expression of KAP1 (www.proteinatlas.org/), suggests that only a subpopulation of KAP1 will be engaged in a stoichiometric interaction with EXO1. While KAP1 has also important roles in transcription, the subpopulation of KAP1 molecules modulating chromatin relaxation in response to DNA damage, a function that is under the strict control of PTMs, will likely be engaged in a stoichiometric interaction with EXO1. We speculate that constitutive physical interaction with a chromatin remodeling factor is beneficial to the cell, as it ensures the presence of EXO1 in the vicinity of regions where damage may occur and where, under the control of CtIP and PTMs such as phosphorylation, ubiquitylation and sumoylation, it can be promptly engaged in the repair of DNA in an error-free manner.
