MCM2-7 helicase is required for DNA replication and has been implicated in the maintenance of genomic instability, as more than 90% of MCM complexes are in dormant origins. Decreased levels of MCM2-7 complex on chromatin lead to genomic instability. The Fanconi anemia (FA) pathway, whose proteins are defective in the genetic disorder that entails bone marrow failure, congenital defects, and acute myeloid leukemia, is also important in maintaining genomic instability through the regulation of homologous recombinatorial repair (HRR) of interstrand cross links (ICL). FANCD2 is the central protein in the FA pathway, exhibiting monoubiquitination in response to DNA damage, and has been shown to interact with MCM2-7. Here we show that monoubiquitinated FANCD2 interacts with MCM2-7 and is required for maintenance of MCM2-7 on chromatin upon DNA interstrand crosslinking (ICL) drug mitomycin C treatment. FANCD2-MCM2-7 also interacts with RAD51, an interaction which is decreased in the absence of FANCD2 or with the expression of the ubiquitination mutant FANCD2(K561R). Also, RAD51 chromatin localization and homologous recombination (HR) function is impaired in MCM7 knockdown cells. MCM2, MCM5, or MCM7 knock down cells exhibit hypersensitive to ICL and G2/M accumulation as well, and the hypersensitivity of MCM7 knock down cells can be rescued by re-expressing MCM7. Importantly FANCD2 and MCM7 are epistatic for MMC hypersensitivity. At the levels of MCM7 knock down, we observe that cells are not defective for replication as measured by BrdU labeling and are not hypersensitive to replication inhibitor hydroxyurea (HU) but remain ICL sensitive. Using recombinant protein reconstitution in vitro, we observe MCM2-7 and FANCI-FANCD2 dependent RAD51 filament stabilization and strand exchange, which are key steps in HRR. MCM2-7 proteins defective for helicase activity and thus replication defective remain functional in these in vitro biochemical assays. On the other hand, a FANCI DNA binding mutant completely abrogates these activities, suggesting that the FANCD2-FANCI-MCM2-7-RAD51 complex requires a regulated interaction with DNA and is separable from MCM2-7 replication function. Taken together, the FA pathway, MCM2-7, and RAD51 work cooperatively in the biochemical and cell response to DNA damage ICL.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.