BRCA1 promotes DNA repair through interactions with multiple proteins, including CtIP and FANCJ (also known as BRIP1/BACH1). While CtIP facilitates DNA end resection when de-acetylated, the function of FANCJ in repair processing is less well defined. Here, we report that FANCJ is also acetylated. Preventing FANCJ acetylation at lysine 1249 does not interfere with the ability of cells to survive DNA interstrand crosslinks (ICLs). However, resistance is achieved with reduced reliance on recombination. Mechanistically, FANCJ acetylation facilitates DNA end processing required for repair and checkpoint signaling. This conclusion was based on the finding that FANCJ and its acetylation were required for robust RPA foci formation, RPA phosphorylation, and Rad51 foci formation in response to camptothecin (CPT). Furthermore, both preventing and mimicking FANCJ acetylation at lysine 1249 disrupts FANCJ function in checkpoint maintenance. Thus, we propose that the dynamic regulation of FANCJ acetylation is critical for robust DNA damage response, recombination-based processing, and ultimately checkpoint maintenance.
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To identify the FANCJ acetylation site(s), myc-tagged C-terminal FANCJ truncation mutants were co-transfected with CBP into 293T cells. By Immunoblot analysis using the pan-acetyl antibody, we found that acetylation of FANCJ required amino acids 1239 to 1249 (Figure 2A, 2C). Consistent with this region being modified, a C-terminal domain of FANCJ similar to a C-terminal p53 control was acetylated in vitro by a HAT-domain protein (Figure 2B, 2C). To determine, which of three lysine (K) residues in this C-terminal region were required for acetylation, we generated three independent FANCJ mutant constructs that converted lysines 1240, 1242, or 1249 to arginine (R). Further transfection experiments revealed that the K1249 was the dominant site for FANCJ acetylation, a lysine that is not conserved in chicken or C. elegans FANCJ species (Figure 2D, 2E).
Next, we sought to provide more conclusive evidence that CBP-induced acetylation on FANCJ was at the K1249 site. We purified FANCJ from 293T cells transfected with a C-terminal myc-tagged FANCJWT or the FANCJK1249R mutant species by immunoprecipitation using a myc antibody. Isolated proteins were then digested with trypsin and subjected to tandem mass spectrometry analysis (LC-MS/MS). FANCJ-derived peptides covering the entire sequence were analyzed, and acetylation sites were identified using MASCOT search algorithm. Most of the acetylated lysine residues were detected in overlapping peptides derived from at least two independent protein preparations. In the FANCJWT, one of these sites was K1249 (Figure 2F). Interestingly, even though by antibody detection, the FANCJK1249R mutant scores unmodified as in Figure 2D; FANCJWT and FANCJK1249R mutant had three additional acetylation marks detected by mass spectrometry (Figure S1). Furthermore, the K1249R mutant had five additional acetylated lysines not found in wild-type FANCJ, suggesting that these sites are not available when K1249 is acetylated (Figure S1). Thus, immunoblot and mass spectrometry analysis confirm that the very last amino acid of FANCJ, lysine 1249 is acetylated.
Given that DNA damage reduces CtIP acetylation [16], we addressed whether DNA damage could alter FANCJ acetylation. Endogenous FANCJ acetylation was enhanced in MCF7 cells treated with zeocin, camptothecin (CPT), or hydroxyurea (HU) as compared to ultraviolet radiation (UV), MMC, or methyl methanesulfonate (MMS) at the dose and time-post treatment analyzed (Figure 3A). Notably, zeocin had a more robust induction of FANCJ acetylation despite the dose of zeocin, CPT, or UV having similar affect on cell survival (Figure S2; data not shown). As found previously, DNA damage did not measurably alter FANCJ co-precipitation with BRCA1 with the exception of UV damage, which could reflect the UV-induced BRCA1 degradation [11], [18] (Figure 3A). DNA damage also induced FANCJ acetylation in HeLa cells, in response to not only CPT, but also MMC (Figure 3A). In response to DNA damage, we also noted that FANCJ protein levels were sometimes enhanced (Figure 3A). To clarify whether acetylation or our ability to detect acetylation was induced by DNA damage, we sought to induce DNA damage in cells in which our ability to detect FANCJ acetylation was not limiting. Indeed, the amount of acetylation on similar levels of exogenous FANCJWT achieved with low dose CBP expression was considerably enhanced following treatment with zeocin or CPT (Figure 3B, 3C). Interactions with BRCA1 and MLH1 were not required for the CBP-induced acetylation of FANCJ, because BRCA1- and MLH1-interaction-defective mutants, FANCJS990A and FANCJK141/142A were readily modified (data not shown). In contrast, following treatment with CPT, acetylation was not detected on the FANCJK1249R mutant (Figure 3C), indicating that DNA damage-induced FANCJ acetylation requires the C-terminal K1249 residue. It remains to be determined, however if FANCJ acetylation is induced by a distinct type of DNA damage.
A. Endogenous FANCJ is acetylated in response DNA damage. MCF7 and HeLa cells were left untreated (UT) or treated with zeocin (6.25 µg/ml for 1 h), MMC (250 nM for 1 h), UV (30 J/m2), MMS (300 µg/ml for 4 h), HU (1 mM for 24 h), or CPT (1 µM for 1 h). Cell lysates were collected at distinct times post damage (zeocin 24 h, MMC 24 h or as indicated, UV 6 h, CPT 24 h, MMS 4 h, and HU 24 h) and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies. B. Exogenous FANCJ is acetylated on lysine 1249 in response to DNA damage. Myc-tagged FANCJ wild-type or mutant species and CBP were co-transfected into 293T cells and left untreated (UT) or treated with zeocin (12.5 µg/ml for 1 h or C. CPT (1 µM for 1 h). Cells were processed at different time points post DNA damage and analyzed for expression and/or acetylation following immunoprecipitation with the indicated antibodies.
The enhanced FANCJ acetylation following DNA damage led us to hypothesize that this modification facilitated FANCJ function in DNA repair. To address this possibility, we made use of this lysine to arginine FANCJK1249R mutant that prevents acetylation and also generated a lysine to glutamine FANCJK1249Q mutant to structurally mimic acetylation. Consistent with these mutants being functional, the purified recombinant proteins displayed similar catalytic activities as FANCJWT (Figure S3). In addition, they were expressed at similar levels as FANCJWT in FANCJ-null FA-J cells (Figure 4A). Similar to FANCJWT, FANCJK1249R and FANCJK1249Q precipitated with known FANCJ interacting partners, BRCA1 and MLH1 [6], [8] (Figure 4B). In addition, the mutants co-localized with BRCA1 in response to DNA damage and the FA-J cells expressing FANCJWT or mutants had similar asynchronous cell cycle profiles (Figure 4C, 4D). The acetylation mutants also restored MMC resistance and the ability of FA-J cells to exit from an abnormal G2/M accumulation, albeit in a manner slightly more robust than FANCJWT (Figure 4E, 4F). Together, these findings suggested that the mutants were enzyme active and functional in vivo; however the mechanism by which the FANCJ mutants restore ICL resistance could be distinct from FANCJWT.
A. The acetylation mutants are expressed in FA-J cells. FA-J cells were complemented with vector, FANCJWT, FANCJK1249R, or FANCJK1249Q. The FA-J cell lines were collected and analyzed or B. lysates were immunoprecipitated with FANCJ antibodies and immunoblot was performed with the indicated antibodies. C. The acetylation mutants localize in nuclear foci of FA-J cells. The FA-J cell lines were seeded onto 6-well plates and incubated overnight. The cells were treated with 1 mM HU and 24 h later immunoflourescence was performed with the indicated antibodies. D. The FA-J cell lines have similar cell cycle profiles. The FA-J cells lines were collected and analyzed by FACS to determine the percentage of cells with 2N and 4N DNA content. E. Expression of acetylation mutants restores MMC resistance. The FA-J cell lines were seed onto 6 well plates and incubated overnight. The cells were either left untreated or treated with increasing doses of MMC. Cells were counted 8 days later and percent survival was calculated. Data represent mean percent s.d. of survival from three independent experiments. F. Expression of acetylation mutants restores G2/M checkpoint exit. The FA-J cell lines were untreated or treated with 0.25 µg/ml melphalan, collected at the indicated times, and analyzed by FACS to determine the percentage of cells in G2/M. Data represent mean percent s.d. of survival from three independent experiments.
Previously, complementation of FA-J cells with a BRCA1-binding defective mutant, FANCJS990A gave the semblance of FANCJWT function. In particular, MMC resistance was restored [8]. However, in contrast to FANCJWT, FANCJS990A provides resistance to MMC by a mechanism dependent on the DNA damage tolerance pathway. Within this tolerance pathway, translesion synthesis polymerases can bypass DNA lesions such as unhooked ICLs and intra-strand crosslinks generated by UV, but not DSBs generated by zeocin. Evidence that FANCJS990A skewed lesion processing towards DNA damage tolerance was based on several findings. First, the sensitivity to MMC in these cells was restored upon depletion of the essential tolerance factor, Rad18 or the translesion polymerase polη, but not upon depletion of the HR protein, Rad54. Second, in comparison to FANCJWT, cells expressing FANCJS990A were hyper-resistant to UV, a phenotype that was reversed upon polη-depletion. Third, in comparison to FANCJWT, FANCJS990A-expressing cells were sensitive to zeocin, indicating reduced DSBR [9]. Thus, we sought to determine whether similar to the BRCA1-binding mutant, the acetylation mutants also functioned differently from FANCJWT. To test this idea, the FA-J cell lines were left untreated or treated with increasing doses of MMC, zeocin, or UV. In comparison to the other FA-J cell lines, the FA-J cell line expressing the acetylation mutant FANCJK1249R was hyper-resistant to UV, but unable to restore normal levels of zeocin resistance. In contrast, the FA-J cell line expressing the acetylation mimic FANCJK1249Q displayed greater resistance to zeocin (Figure 5A; Figure S2). Thus, in response to UV and zeocin, cells expressing the acetylation mutants are distinct from each other as well as from cells expressing FANCJWT. 2ff7e9595c
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