Although 15 FA proteins have been identified to date that participate in a common pathway to repair ICLs, much still remains unknown about the detection and processing of these deleterious lesions. In this study, we identify two novel FANCD2-binding partners, MSH2 and MLH1. MSH2 binds to FANCD2-L in response to crosslinking agents, and this interaction requires ATR and is enhanced by the presence of MLH1. MLH1 interacts with FANCD2 only in response to crosslinking agents and requires MSH2. In addition, we find that FANCD2 and FANCI monoubiquitylation and chromatin loading and FANCD2 foci formation are impaired in MSH2-deficient cells, but not in MLH1-deficient cells. However, both MSH2- and MLH1-deficient cells are hypersensitive to multiple crosslinking agents and display the characteristic increase in radial formation common to all FA mutant cells. Lastly, studies in both human cell lines and Drosophila
mutants indicate an epistatic relationship between FANCD2, MSH2 and MLH1 in ICL repair. Although previous studies have demonstrated interactions between MMR and FA or FA-associated proteins (25
), this is the first demonstration of a physical and functional interaction between the pivotal FA protein FANCD2 with MMR proteins (MSH2 and MLH1), and also the first report of an interaction between MSH2 and an FA protein.
Previous studies have implicated human MutS complexes in the recognition and early processing of ICLs. MutS complexes are able to bind ICLs in vitro
), and the initial processing steps of an ICL require MSH2 (22
), but not MLH1. Evidence has also been presented that MSH2 plays a role in ATR activation and recruitment to sites of DNA damage (19
). Interestingly, MSH2 has also been shown to interact with ERCC1-XPF in response to ICLs induced by CDDP (42
), and ERCC1-XPF has been proposed to perform the first incision of the crosslink in ICL repair (43
). Taken together with our findings that MSH2 interacts with FANCD2 and is required for the monoubiquitylation and chromatin loading of FANCD2 and FANCI, we therefore suggest that MSH2 plays a crucial role in the detection of ICLs and the early signaling events leading to the activation of the FA pathway. Given that several FA core complex proteins are phosphorylated in an ATR-dependent manner, including FANCA, E, G and M, as well as FANCD2 in response to DNA damage (28
), it is likely that MSH2 initiates a signaling cascade responsible for activation and chromatin loading of the FA core complex in addition to FANCD2 and FANCI as we have shown.
Our data show that MLH1-, MSH3- and MSH2-deficient cells are hypersensitive to DNA crosslinking agents. Previous reports have suggested that MMR-defective cells are resistant to CDDP and psoralen ICLs (48
), but this result has not been consistent. MSH2-deficient cells have been shown by several groups to be hypersensitive to MMC and psoralen ICLs (51
). In addition, PMS2-deficient HeLa cells and MLH1-defective Raji 10 cells have both been shown to be hypersensitive to MMC (52
), supporting a role for the MutLα complex in ICL repair. Even more recently, a study now describes a clinical mutation in MLH1 that causes sensitivity to MMC (53
), again lending credence to our data showing that MMR-defective cells are hypersensitive to DNA crosslinking agents. Lastly, other groups have shown that sensitivity to CDDP in some MMR-defective cells differs based on the p53 status of each subline (54
). In this study, we have shown sensitivity to ICL agents in four different species deficient in one of three MMR proteins.
Other recent reports have shown that FANCJ interacts with the MutLα complex, and that disruption of this interaction results in hypersensitivity to interstrand crosslinking agents (25
). It was therefore suggested that MLH1 may function to facilitate downstream ICL repair through the regulation of FANCJ helicase activity. Here we describe a damage-inducible interaction between FANCD2 and MLH1. As FANCD2 also binds to FANCJ (Chen and Kupfer, unpublished data, 2011), it is conceivable that FANCD2 escorts MLH1 to FANCJ to facilitate the unwinding of DNA and subsequent ICL repair. This would place MLH1 downstream of FANCD2 and FANCI monoubiquitylation and chromatin loading, and would explain the differences we observe between MSH2- and MLH1-deficient cells.
Mutations in MMR genes are most often associated with hereditary non-polyposis colorectal cancer (HNPCC) (56
). Given that FA patients are generally not at a significantly increased risk for this type of cancer, it would be easy to dismiss the importance of the interaction between FA and MMR proteins. However, some cases of colon cancer have been reported in FA patients (57
) and preliminary studies have suggested that some subsets of colorectal cancer patients may have alterations in FA genes (58
). In addition, although monoallelic mutations in MMR genes result in HNPCC, biallelic mutations in MMR genes are now being described as the basis for constitutional mismatch-repair deficiency syndrome (CMMR-D) (59
). Interestingly, similar to FA, CMMR-D is characterized by a predisposition to childhood cancers, mainly hematological malignancies or brain tumors, and café au lait spots (60
). And like CMMR-D, monoallelic mutation of some FA genes results in familial breast cancer, whereas biallelic mutation of these same genes results in FA (61
). CMMR-D has been attributed to biallelic mutations in MSH6, MSH2, MLH1 and PMS2 to date (62
). The overlap between FA and CMMR-D as diseases is indicative of a likely functional overlap between the FA and MMR pathways.
A recent report by Xie et al.
) also shows a possible link between FANCJ and MMR signaling. They demonstrate that a clinical mutation in MLH1, which ablates its ability to bind to FANCJ, causes sensitivity to MMC. However, they also find that FANCJ deficiency results in reduced MMR signaling as measured by a decrease in Chk1 phosphorylation after stimulation with methyl nitrosourea, an agent that induces O6
-methylguanine formation and spurs an MMR response. These data suggest an overlap in function between MMR and FA proteins, and support our data showing that MMR is defective in multiple FA cell lines.
This is the first report of defective MMR in FA cells; however, other DNA repair proteins have been shown to be required for MMR that were previously unappreciated in this respect. Deficiency in MRE11, a protein first implicated in the repair of DNA double-strand breaks, has now been shown to lead to defective MMR and microsatellite instability (67
). Similar to our studies, MRE11 interacts with MLH1, and depletion of MRE11 results in MMR deficiency as measured by an analogous plasmid-based assay. This suggests that perhaps even more DNA repair proteins are involved in the MMR pathway or other functions of MMR proteins remain to be discovered.
Our data suggest a significant physical and functional interaction between the FA and MMR pathways. We therefore propose a 2-fold model (Fig. ) where MSH2 and MLH1 are required for ICL repair. MSH2 is responsible for the activation of the FA pathway, likely through the recognition of the lesion and activation and recruitment of ATR. This model is supported by a recent report showing a requirement for MSH2 in ATR recruitment following CDDP treatment (20
). Downstream of FANCD2 and FANCI monoubiquitylation, MLH1 could be shuttled to FANCJ by FANCD2 in order to facilitate DNA unwinding and subsequent repair steps. Conversely, FA proteins are involved in MMR, where at least FANCA and FANCD2 enhance the interaction between MSH2 and MLH1.
Figure 8. A model showing the overlapping functions of MMR and FA proteins in ICL and MMR. MSH2 is likely involved in the detection of ICLs and early signaling events leading to the monoubiquitylation and chromatin loading of FANCD2 and FANCI, such as recruitment (more ...)