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The FANCJ/MutLα interaction is required for correction of the cross-link response in FA-J cells

2007; Springer Nature; Volume: 26; Issue: 13 Linguagem: Inglês

10.1038/sj.emboj.7601754

1460-2075

Min Peng, Rachel Litman, Jenny Xie, Sudha Sharma, Robert Brosh, Sharon B. Cantor,

Genetic factors in colorectal cancer

Article21 June 2007free access The FANCJ/MutLα interaction is required for correction of the cross-link response in FA-J cells Min Peng Min Peng Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Rachel Litman Rachel Litman Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Jenny Xie Jenny Xie Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Sudha Sharma Sudha Sharma Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA Search for more papers by this author Robert M Brosh Jr Robert M Brosh Jr Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA Search for more papers by this author Sharon B Cantor Corresponding Author Sharon B Cantor Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Min Peng Min Peng Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Rachel Litman Rachel Litman Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Jenny Xie Jenny Xie Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Sudha Sharma Sudha Sharma Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA Search for more papers by this author Robert M Brosh Jr Robert M Brosh Jr Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA Search for more papers by this author Sharon B Cantor Corresponding Author Sharon B Cantor Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA Search for more papers by this author Author Information Min Peng1,‡, Rachel Litman1,‡, Jenny Xie1, Sudha Sharma2, Robert M Brosh2 and Sharon B Cantor 1 1Department of Cancer Biology, University of Massachusetts Medical School Women's Cancers Program, UMASS Memorial Cancer Center, Worcester, MA, USA 2Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA ‡These authors contributed equally to this work *Corresponding author. Department of Cancer Biology, UMASS Medical School, 364 Plantation Street, LRB 415, Worcester, MA 01605, USA. Tel.: +1 508 856 4421; Fax: +1 508 856 1310; E-mail: [email protected] The EMBO Journal (2007)26:3238-3249https://doi.org/10.1038/sj.emboj.7601754 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info FANCJ also called BACH1/BRIP1 was first linked to hereditary breast cancer through its direct interaction with BRCA1. FANCJ was also recently identified as a Fanconi anemia (FA) gene product, establishing FANCJ as an essential tumor suppressor. Similar to other FA cells, FANCJ-null (FA-J) cells accumulate 4N DNA content in response to DNA interstrand crosslinks (ICLs). This accumulation is corrected by reintroduction of wild-type FANCJ. Here, we show that FANCJ interacts with the mismatch repair complex MutLα, composed of PMS2 and MLH1. Specifically, FANCJ directly interacts with MLH1 independent of BRCA1, through its helicase domain. Genetic studies reveal that FANCJ helicase activity and MLH1 binding, but not BRCA1 binding, are essential to correct the FA-J cells’ ICL-induced 4N DNA accumulation and sensitivity to ICLs. These results suggest that the FANCJ/MutLα interaction, but not FANCJ/BRCA1 interaction, is essential for establishment of a normal ICL-induced response. The functional role of the FANCJ/MutLα complex demonstrates a novel link between FA and MMR, and predicts a broader role for FANCJ in DNA damage signaling independent of BRCA1. Introduction In the absence of DNA repair proteins, cell cycle checkpoints and/or DNA damage repair pathways are not properly activated. This inability to actively respond to DNA damage can lead to massive chromosomal damage and even cell death. In some cases, mutations in DNA repair proteins can contribute to multiple cancer syndromes. Studies on the genetic causes of the cancer-prone syndrome Fanconi anemia (FA) revealed that genetic mutations associated with hereditary breast cancer were also associated with FA. For example, the hereditary breast cancer gene, BRCA2 was shown to be the gene defect in the FA-D1 patient complementation group, revealing that BRCA2 was FANCD1 (Howlett et al, 2002). Likewise, FANCJ (also called BACH1/BRIP1) was identified as the gene defective in the FANCJ-null (FA-J) patient complementation group (Levitus et al, 2005; Levran et al, 2005; Litman et al, 2005), and was initially linked to hereditary breast cancer. This link was based on its direct binding to BRCA1 and through the identification of two breast cancer patients with mutations in FANCJ, which also altered its helicase activity in vitro (Cantor et al, 2004, 2001). This connection was furthered by the finding that FANCJ (BRIP1) mutations confer a two-fold increase in the risk of developing breast cancer (Seal et al, 2006). While other FA genes have not been linked to breast cancer, the network of at least 13 genes (designated FANCA to FANCN) is critical for maintaining chromosomal integrity (Thompson, 2005). Although the molecular function of these proteins is not clear, several gene products, including FANCA, B, C, D, E, F, G, L and M, form a nuclear core complex (the FA core complex), that is required for monoubiquitination of FANCD2. The FA proteins BRCA2/FANCD1, PALB2/FANCN and FANCJ are not required for this event and are considered downstream of FANCD2 monoubiquitination. Nevertheless, all FA proteins contribute to processing interstrand crosslinks (ICLs) (Thompson, 2005). Consequently, in the absence of FA proteins, ICL treatment leads to reduced cell viability and an accumulation of cells with a 4N DNA content representing cells in either late S or G2/M. This ICL-induced cell cycle progression defect and sensitivity to ICLs is restored upon reintroduction of the missing FA gene (Dutrillaux et al, 1982; Kaiser et al, 1982; Kupfer and D'Andrea, 1996; Kupfer et al, 1997; Heinrich et al, 1998; Sala-Trepat et al, 2000; Chandra et al, 2005). However, the FA-related function or associated partners required for a proper ICL response is not known. Consistent with other FA cells, FA-J cells have an ICL-induced cell cycle progression defect that can be corrected upon re-introduction of wild-type (WT) FANCJ cDNA (Litman et al, 2005). This cell cycle progression defect has also been described as a prolonged G2/M arrest (Miglierina et al, 1991) or 4N DNA content accumulation (Akkari et al, 2001). The cause of this ICL response in FA cells is not presently understood, but is thought to involve delayed repair and/or failure to restart replication (Thompson et al, 2005). Unlike the majority of FA proteins, FANCJ has defined domains. Specifically, FANCJ binds directly to BRCA1 (Cantor et al, 2001) and is a DNA helicase (Cantor et al, 2004). Determining the importance of these domains could further our understanding of how FA proteins function in an ICL-induced response. Attempts to define the functions of FANCJ domains in the ICL response have been limited to chicken DT40 cells, where the FANCJ/BRCA1 interaction is not conserved (Bridge et al, 2005). If FANCJ operates independent of BRCA1 for a particular ICL response function, a remaining question will be whether FANCJ forms a complex with other proteins independent of BRCA1 to perform that function. Here, we investigated whether FANCJ helicase activity or the FANCJ interaction with two distinct proteins was required for restoring FANCJ's ICL response. Specifically, we identified that FANCJ interacts with the MutLα mismatch repair complex, independent of BRCA1. Our findings demonstrate for the first time that the FANCJ/MLH1 interaction is as critical as FANCJ helicase activity for restoring a normal cell cycle progression and resistance of FA-J cells to ICLs. In contrast, the FANCJ/BRCA1 interaction is dispensable for normalizing the response of FA-J cells to ICLs, suggesting that FANCJ functions in distinct complexes to facilitate multifaceted DNA repair functions. Results FANCJ functions independently of BRCA1 to correct FA-J cells We had previously shown that introduction of WT FANCJ cDNA into FA-J cells corrects the ICL-induced cell cycle progression defect (Litman et al, 2005). However, it is unclear how FANCJ contributes to the ICL response to restore the FA pathway, especially given that FANCJ's role in the FA pathway appears to be independent of BRCA1, at least in chicken cells (Bridge et al, 2005). To verify and extend this finding, we addressed whether FANCJ binding to BRCA1 was required to correct the ICL-induced cell cycle progression defect in FA-J cells. We reconstituted FA-J cells with vector, WT, or the S990A FANCJ construct that is ablated for BRCA1 binding (Yu et al, 2003) (Figure 1C). Both WT and S990A versions of FANCJ corrected the ICL-induced cell cycle progression defect observed in FA-J cells compared to vector alone (Figure 1A and B). These data support the finding that FANCJ operates independent of BRCA1 to correct FA-J cells. Figure 1.The FANCJ/BRCA1 interaction is dispensable for correction of the 4N DNA accumulation defect in FA-J cells. (A) FA-J cells were reconstituted with vector, WT or S990A, and FANCJ expression was analyzed in whole-cell extracts (WCE) by Western blot. β-Actin served as a loading control for the WCE samples. (B) Immunoprecipitations with FANCJ (E67) were analyzed by Western blot with the indicated Abs. (C) FA-J cells reconstituted with vector, WT, or S990A FANCJ were either left untreated or treated with melphalan, and the percentage of cells with 4N DNA content was analyzed by FACS. The percentage of cells with 4N DNA content after ICL-treatment was averaged for each cell line from four independent experiments, with standard deviation (s.d.) indicated by error bars. Download figure Download PowerPoint FANCJ is physically linked to the MutLα complex Since FANCJ binding with BRCA1 was not required to correct the ICL-induced cell cycle progression defect in FA-J cells, we set out to identify additional FANCJ interacting partners that may function with FANCJ in this ICL-induced response. A WT double tagged FANCJ construct was used to create a stable line of HeLa S3 cells. Using a two-step immunoaffinity strategy, the double tagged FANCJ was sequentially immunopurified (Nakatani and Ogryzko, 2003). Interacting proteins co-purifying with the double tagged WT FANCJ were eluted and visualized by silver stain. FANCJ migrated at the expected ∼140 kDa size (Figure 2A). Individual bands were excised from the gel and analyzed by mass spectrometry (LC-MS/MS). As expected, FANCJ copurified with BRCA1 that was identified as the 250 kDa band. Unique partners were identified, including the MMR proteins, MLH1 and PMS2, which form the MutLα heterodimer (Schofield and Hsieh, 2003) (Figure 2A). Western blot analyses using specific antibodies (Abs) confirmed the presence of these proteins (Figure 2B). To determine whether the MutLα complex associated with the native FANCJ protein, MCF7 cell extracts were immunoprecipitated (IP) with FANCJ Abs E67 and E47, and the presence of co-precipitating MLH1, PMS2 and BRCA1 proteins was evaluated by Western blot (Figure 2C). While FANCJ Ab precipitated the MutLα complex in the MCF7 cells, a MutLα complex was not precipitated with preimmune Abs (PI) or FANCJ Abs, in 293T cells, which lack expression of the MutLα complex (Trojan et al, 2002). Moreover, FANCJ was not precipitated with the MLH1 Ab in FA-J cells, which lack expression of FANCJ, unless FANCJ was reintroduced (Figure 2D). In contrast, a FANCJ/MLH1 interaction was readily detected in other FA cell lines, irrespective of gene correction, such as FA-A, FA-D1 and FA-D2 (Supplementary Figure 1). Furthermore, the interaction between FANCJ and the MutLα complex was stable in HeLa cells in the presence or absence of DNA damage (Figure 2E). Figure 2.FANCJ interacts with the MMR proteins MLH1 and PMS2. (A) Silver-stained gel of the WT FANCJ (F) compared to vector (V)-purified complexes from HeLa S3 cells by consecutive Flag and HA purification steps (Flag/HA). Identified unique bands are indicated and FANCJ is observed as two species, the 140 kDa band is labeled. (B) Western blot detection of Flag/HA-purified FANCJ complexes. (C) Immunoprecipitations with either FANCJ (E67 or E47) or MLH1 Abs from MCF7 or 293T cells were analyzed by Western blot with the indicated Abs. (D) Western blot shows the presence of the indicated proteins from MLH1 IPs from FA-J cells reconstituted with vector or WT FANCJ. (E) HeLa cells were either left untreated or treated with 1 mM HU for 24 h or 2.4 μg/ml MMC for 1 h. HeLa cell lysates were IPed with PI or FANCJ Abs followed by Western blot analysis with the indicated Abs. Download figure Download PowerPoint The helicase domain of FANCJ binds directly to MLH1 independent of BRCA1 MLH1 was previously reported to be part of a BRCA1 complex (Wang et al, 2000; Greenberg et al, 2006); therefore, we examined whether BRCA1 mediated the interaction between FANCJ and the MutLα complex. First, we noted that unlike FANCJ, BRCA1 was not readily detected in an MLH1 precipitation (Figure 2C). Next, we addressed whether FANCJ precipitated with the MutLα complex in BRCA1-deficient cells. Expression of BRCA1 was stably suppressed in MCF7 cells by an shRNA vector, as previously demonstrated (Litman et al, 2005). In cells expressing both a control shRNA specific to eGFP or an shRNA specific to BRCA1, FANCJ Abs efficiently co-precipitated the two components of the MutLα complex (Figure 3A), suggesting that FANCJ binds the MutLα complex independent of BRCA1. In support of this finding, the helicase domain of FANCJ was required for MLH1 binding, while a C-terminal region of FANCJ was required for BRCA1 binding (Figure 3B). To further assess the nature of the FANCJ/MLH1 interaction, we incubated recombinant FANCJ or BRCA1 with MLH1 that had been translated in vitro. MLH1 and FANCJ were precipitated by their corresponding Abs, and their interactions were analyzed by Western blot. FANCJ and MLH1 proteins were co-precipitated with both FANCJ and MLH1 IPs, whereas BRCA1 was robustly precipitated only in the FANCJ IP (Figure 3C). A direct interaction between FANCJ and MLH1 was confirmed by ELISA assay using purified recombinant proteins. FANCJ bound MLH1 in a protein concentration-dependent manner (Figure 3D). Furthermore, the interaction of FANCJ and MLH1 was demonstrated to be DNA independent, as evidenced by the similar colorimetric signal observed for FANCJ/MLH1 interaction in the presence of ethidium bromide (EtBr) or DNaseI (Figure 3E). These results suggest that FANCJ makes direct contacts with MLH1, independent of BRCA1 or PMS2. Figure 3.FANCJ helicase domain associates with the MutLα complex independent of BRCA1 and through a direct interaction with MLH1. (A) MCF7 cells were stably infected with a lentivirus encoding shRNA for either eGFP or BRCA1. FANCJ IP was performed followed by Western blot for the indicated proteins. (B) MCF7 cells were transiently transfected with pCDNA3 vectors containing no insert (−), full-length FANCJ (FL), helicase domain including amino-acid residues 1–882 (HD) or C-terminus including residues 882–1249 (CT) of FANCJ, and then IPed with the Myc Ab (9E10). Arrows indicate the respective FANCJ myc-tagged species. Immunoglobulin (IgG) is shown. (C) Western blot of the indicated IP experiments in which in vitro translated MLH1 was incubated with recombinant FANCJ or BRCA1 proteins. (D) Purified recombinant MLH1 or BSA was coated onto ELISA plates. Following blocking with 3% BSA, the wells were incubated with increasing concentrations of purified recombinant FANCJ (0–40 nM) for 1 h at 30°C, and bound FANCJ was detected by ELISA using a rabbit polyclonal Ab against FANCJ, followed by incubation with secondary horseradish peroxidase (HRP)-labeled Abs and OPD substrate. Data points are the mean of three independent experiments performed in duplicate, with s.d. indicated by error bars. (E) ELISA was performed as described in panel D using 4.9 nM FANCJ alone or in the presence of EtBr (50 μg/ml) or DNaseI (2 μg/ml). BSA (3%) was used as a control instead of MLH1 during the coating step. Download figure Download PowerPoint PMS2 contributes to the FANCJ/MLH1 interaction in vivo Given that MLH1 forms a heterodimer with PMS2, we next assessed whether PMS2 binding to MLH1 contributed to the MLH1/FANCJ interaction in vivo. To address this possibility, we tested the ability of different MLH1 constructs to precipitate FANCJ in the absence or presence of PMS2. WT full-length MLH1 and several MLH1-myc fusion proteins of varying length were generated and transiently transfected into MutLα-null 293T cells. To determine which of these MLH1 fragments were expressed and/or co-IPed FANCJ, MLH1 was precipitated from cell lysates with either myc or MLH1 Abs. While cotransfecting PMS2 with MLH1 did not alter the expression of MLH1, the ability of FANCJ to form a complex with MLH1 was enhanced. With the addition of PMS2, FANCJ precipitated with the MLH1 constructs N2, C2, and C3, which in the absence of PMS2 had failed to precipitate FANCJ (Figure 4A). Thus, in the presence of PMS2, only one of the two MLH1-FANCJ interacting domains (478–508) (D1) or (736–744) (D2) was required (see Figure 4C), suggesting that PMS2 facilitates the MLH1/FANCJ interaction. PMS2 stability is dependent on the MLH1 C-terminus (Mohd et al, 2006); not surprisingly, we found that ablation of a C-terminal region of MLH1 (703–725) reduced both PMS2 and FANCJ binding (Figure 4B). Figure 4.PMS2 facilitates the FANCJ interaction with the MLH1 C-terminus. (A) MLH1 or Myc (9E10) IP experiments were performed from 293T cells that were transfected with vector alone (V), full-length MLH1 (WT) or MLH1 species, alone or in combination with PMS2 (C1–C3, N1, N2). IP products were analyzed by Western blot with FANCJ, PMS2, and MLH1 Abs. (B) MLH1 IP experiments were performed from 293T cells that were transfected with vector alone (V), full-length MLH1 (WT) or MLH1 species, in combination with PMS2 (C4–C7). IP products were analyzed by Western blot with FANCJ, PMS2, and MLH1 Abs. (C) Schematic representation of the MLH1/FANCJ dimer domains (D1, D2), and the region between 703–725 is highlighted as an essential element for maintaining the MLH1/PMS2/FANCJ complex. Download figure Download PowerPoint MutLα functions downstream of FANCD2 monoubiquitination To appreciate the physiological significance of a FANCJ/MutLα interaction, we next, addressed whether the MutLα complex functioned with FANCJ in the FA pathway. We had previously shown that in FANCJ-deficient cells, DNA damage-induced FANCD2 monoubuiquitination was intact (Litman et al, 2005). Similarly, we found that incubation of MutLα-deficient cells (HCT116 and HEC-1A) with hydroxyurea (HU) leads to efficient FANCD2 monoubiquitination (Supplementary Figure 2A), suggesting that similar to FANCJ, MutLα functions downstream of FANCD2. Given that suppression of MMR proteins has been reported to reduce the survival of cells upon ICL-treatment, (Aquilina et al, 1998; Fiumicino et al, 2000), we next asked whether similar to FANCJ deficiency, MutLα deficiency also sensitizes cells to ICLs. First, we suppressed MutLα using siRNA reagents in MCF7 cells versus a luciferase control. Second, we reconstituted HCT116 cells null for MutLα with vector or MutLα expressing cDNAs. In both experiments, there was no measurable change in ICL sensitivity, in the presence or absence of MutLα expression (Supplementary Figure 2B and data not shown). Given that MMR proteins bind and process ICLs (Duckett et al, 1996; Yamada et al, 1997; Zhang et al, 2002), activate multiple DNA damage-induced checkpoints, such as intra S and G2/M (4N) arrest (Brown et al, 2003; Cejka et al, 2003), and participate in the repair of ICLs by promoting recombination (Zheng et al, 2006), we considered that MutLα suppression could bypass ICL sensitivity through loss of checkpoint (Cejka et al, 2003), and/or by activating default non-recombination-based repair pathways, as reported (Zheng et al, 2006). Thus, we considered that to unmask function of MutLα in the ICL response with FANCJ, it would be necessary to selectively ablate the MLH1/FANCJ interaction, while maintaining other MLH1 functional interactions (i.e. PMS2 binding). Disruption of the native MLH1/FANCJ interaction generates ICL sensitivity To define the domain on FANCJ required for MLH1 binding, we generated several FANCJ-myc fusion proteins of varying length and expressed them in MCF7 cells (Figure 5A and E). To determine which of these FANCJ fragments were expressed and/or co-IPed MLH1, FANCJ was precipitated from cell lysates with myc Abs. Full-length FANCJ and FANCJ expression constructs including the FANCJ N-terminal amino-acid residues 1–145 precipitated MLH1 (Figure 5A and C). These results suggested that FANCJ N-terminal residues 1–145 were required for binding to MLH1. To assess whether residues in this region were sufficient for MLH1 binding, we inserted FANCJ residues 128–158 within the eGFP gene sequence to create an eGFP-fusion protein. In contrast to eGFP alone, the eGFP-FANCJ fusion protein readily co-precipitated MLH1 (Figure 5B), suggesting that FANCJ 128–158 was sufficient for MLH1 binding. Furthermore, expression of the eGFP-FANCJ fusion protein in cells perturbed the formation of the native FANCJ/MLH1 interaction, as determined by both FANCJ and MLH1 IP and Western blot experiments (Figure 5D), confirming that this region of FANCJ was essential for mediating the MLH1 interaction. Figure 5.Expression of FANCJ residues 128–158 disrupts the FANCJ/MLH1 interaction to generate ICL sensitivity. (A) Myc (9E10) IP experiments were performed from MCF7 cells that were transfected with vector alone (−), full-length FANCJ (FL) and the different FANCJ constructs (A–G) shown in panel C, followed by Western blot with MLH1, and Myc Abs. The asterisk denotes the migration of the different myc-tagged FANCJ species. (B, D) Myc IP experiments were performed from MCF7 cells that were transfected with either eGFP empty vector or the 128–158 FANCJ-eGFP constructs, followed by Western blot with the indicated Abs. (C) The different FANCJ constructs are indicated with a positive (+) or negative (−) to indicate binding to MLH1. (E) MCF7 cells transfected with vector alone or the 128–158 FANCJ-eGFP construct, treated with increasing concentrations of MMC and incubated for 4–5 days. Cell growth was measured by ATP content. Three independent representative experiments are shown and depicted by lines with squares, triangles, and diamonds. Solid lines represent cells transfected with empty-eGFP vector and hatched lines represent cells transfected with 128–158-eGFP vector (for color figure see online version). Download figure Download PowerPoint Next, we addressed whether expression of the 128–158 FANCJ-eGFP fusion protein and the resulting perturbation of the native FANCJ/MLH1 interaction would render cells sensitive to ICLs. MCF7 cells were transfected with vectors expressing either the 128–158 FANCJ-eGFP fusion protein or eGFP alone, plated and treated with increasing concentrations of Mitomycin C (MMC). The overall trend upon expression of the 128–158 FANCJ-eGFP fusion protein was reduced cellular survival compared to expression of the eGFP control, despite some variability between experiments (Figure 5E). While the enhanced sensitivity was consistent with the possibility that a FANCJ/MLH1 interaction was required for ICL repair, we considered that binding of the fusion protein to MLH1 might have altered additional MLH1 functions not specific to FANCJ. Thus, we sought to identify a method to ablate the FANCJ/MLH1 interaction without altering native MLH1 protein or being reliant on transfection efficiency to disrupt the native FANCJ/MLH1 interaction. Lysines 141 and 142 of FANCJ are required for the FANCJ/MLH1 interaction Given that mutational analysis revealed that FANCJ co-precipitated with MLH1, except when FANCJ residues 140–145 were absent (Figure 5A and C), we assessed the importance of these residues for binding MLH1 within the context of the full-length FANCJ protein. Thus, we generated three independent FANCJ mutant constructs that converted lysine 141 and 142 to alanine (K141/142A), glutamine 143 to a glutamic acid (Q143E), or serine 145 to an alanine (S145A). While the WT FANCJ and all three mutant versions were expressed and efficiently co-precipitated BRCA1, the K141/142A version demonstrated a dramatic reduction in the co-precipitation of MLH1 (Figure 6A), suggesting that these two lysines were required for MLH1 binding. Figure 6.MLH1 binding to FANCJ is essential to correct FA-J cells. (A) Myc IP experiments were performed from MCF7 cells that were transfected with vector alone (−), FL, V, Q143E, S145A, and K141/142A FANCJ constructs, followed by Western blot with the indicated Abs. FA-J cells were reconstituted with empty vector, WT, K141/142A, or K52R FANCJ vectors, and FANCJ expression was analyzed by whole-cell extracts; β-actin served as a loading control for the WCE samples. Western blot shows the presence of the indicated proteins from FANCJ IPs from FA-J cells reconstituted with vector, WT, or K141/142A FANCJ. (B) FA-J cell lines reconstituted with empty vector, WT, K141/142A, or K52R FANCJ were either left untreated or treated with melphalan. The percentage of cells with 4N DNA content after ICL treatment was averaged for each cell line from four independent experiments, with standard deviation (s.d.) indicated by error bars. (C) FA-J cells reconstituted with vector, WT, K141/142A, K52R, or S990A FANCJ were seeded on 24-well plates and incubated overnight under normal growth conditions. The cells were then treated with the indicated doses of MMC and incubated for 8 days. On the final day, the cells were counted and the percentage of live cells was calculated. Experiments were performed in triplicate and a representative plot is shown. (D) The IC50 dose for the FA-J vector (250 nM) was compared for all mutants, and error bars represent the standard deviation. Download figure Download PowerPoint We considered that the K141/142A mutation in FANCJ could have not only disrupted MLH1 binding, but also FANCJ helicase activity. Thus, we generated recombinant versions of WT (Cantor et al, 2004) and the K141/142A FANCJ proteins to assess whether this mutant version was enzymatically active. The recombinant K141/142 FANCJ protein was detected as a single Coomassie-stained band analyzed by SDS–PAGE, that co-migrated with the WT FANCJ recombinant protein (Supplementary Figure 3A and D). The DNA unwinding activity of K141/142A FANCJ on a forked duplex DNA substrate was compared to unwinding activity of WT FANCJ. Both K141/142A FANCJ and WT FANCJ were found to be proficient in unwinding, whereas K52R FANCJ, as previously demonstrated, failed to unwind the forked duplex substrate (Gupta et al, 2005) (Supplementary Figure 3B). Furthermore, K141/142A FANCJ and WT FANCJ unwound the forked duplex substrate in a protein concentration-dependent manner, achieving 90% of unwound substrate at the highest helicase concentration tested (Supplementary Figure 3C). Thus, the K141/142 mutant only disrupts MLH1 binding, but not FANCJ helicase activity. FANCJ function depends on MLH1 binding to correct FA-J cells Next, we tested the ability of K141/142A FANCJ cDNA to correct the cell cycle progression defect in FA-J cells. We used retroviral infection to stably infect FA-J cells with cDNA encoding the vector, WT, K141/142A or K52R Flag/HA-tagged FANCJ constructs, which expressed similarly (Figure 6A). Moreover, an MLH1 IP demonstrated that the MLH1/PMS2 complex was intact in FA-J cells and was able to precipitate the reconstituted FANCJ (Figure 2D). As in MCF7 cells, in FA-J cells MLH1 co-IPed with WT FANCJ, but was dramatically reduced with the K141/142A mutant FANCJ (Figure 6A). FA-J cells containing vector, WT, K52R, or K141/142A FANCJ were treated with melphalan to induce ICLs, as described (Litman et al, 2005). The proportion of vector containing FA-J cells with 4N DNA content increased after melphalan treatment, similar to previous experiments. As before, the proportion of WT FANCJ containing FA-J cells with 4N DNA content (∼30%) was about half that of vector containing cells (∼70%). We found that cells containing the catalytically inactive FANCJ helicase (K52R) failed to correct the 4N accumulation defect (∼66%). Likewise, the proportion of K141/142A FANCJ containing FA-J cells with 4N DNA content (∼68%) resembled that of vector containing FA-J cel