Abstract
Fanconi anemia (FA) is an inherited hematological disorder characterized by bone marrow failure, birth defects, and cancer susceptibility, typically leading to squamous cell carcinomas and acute myelogenous leukemia. Twelve FA genes have been described, eight of which function together in a multiprotein, upstream “FA core complex” to mediate the S-phase and DNA damage-induced monoubiquitylation of two downstream proteins, FANCD2 and FANCI. Despite this knowledge the precise function of the FA proteins is not well understood because they function as part of a network of proteins that have not been completely defined.
Recently we developed a new animal model for FA research using extracts from the eggs of Xenopus laevis. Xenopus extracts are cell cycle synchronized and contain nuclear proteins that are stockpiled for DNA replication. We showed that FA gene orthologs (xFA), like their human counterparts, form complexes that are required for the monoubiquitylation of xFANCD2 in response to DNA damage. Xenopus laevis extracts are thus a powerful system to analyze the endogenous state of xFA protein complexes and their components in an S phase, replication-competent context.
The objective of this study was to isolate protein complexes containing the xFA core complex protein, xFANCM and xFANCM-interacting proteins.
Using a co-immunoprecipitation approach followed by mass spectrometry, we identified a novel protein-binding partner of xFANCM (termed xMIP-1, for xFANCM Interacting Protein 1). The interaction was confirmed by reciprocal coimmunoprecipitation in both Xenopus extracts and human cells. Surprisingly, co-fractionation demonstrated that xFANCM was present in two protein complexes during S phase; one containing FA core complex members (<900 kDa) as expected, and another previously undescribed complex (>900 kDa) containing xMIP-1. Because xMIP-1 is a partner protein of xFANCM we wanted to determine if xMIP-1, like xFANCM, was required for the monoubiquitylation of xFANCD2. This was done using a DNA stimulation assay, where upon immunodepletion of xMIP-1 from egg extracts, we were able to observe the monoubiquitylation of xFANCD2 in response to DNA structures as a size shift via immunoblot. The absence of xMIP-1 had no detectable effect on the monoubiquitylation of xFANCD2 suggesting that xMIP-1, unlike xFANCM, was not required for xFANCD2 monoubiquitylation. To explore a functional link between xFANCM and xMIP-1 we used egg extracts to show that xMIP-1, like xFANCM, was recruited to replicating chromatin and exhibited a size shift during the replication process. Furthermore immunodepletion of xFANCM from egg extracts reduced recruitment of xMIP-1 to replicating chromatin, suggesting that xMIP-1 chromatin binding was dependent on xFANCM. In contrast, xMIP-1 recruitment to replicating chromatin was not affected by the immunodepletion of other FA core complex proteins tested, suggesting that xMIP-1 chromatin binding is independent of the FA core complex. To further characterize the observed DNA binding activity of xMIP-1 we used the DNA stimulation assay and several defined DNA structures. Surprisingly xMIP-1 showed a double-stranded DNA stimulated mobility shift similar to those reported previously for xFANCD2 (Sobeck et al., 2007) and xMRE11 (Costanzo et al., 2001) suggesting xMIP-1 may play a role in the DNA damage response.
Our data suggests xFANCM is a member of an S phase complex that has not been previously described with a “non-FA” partner protein that may function with xFANCM during the DNA damage response to maintain genomic stability.
Genome-Wide Dynamic Evaluation of the UV-Induced DNA Damage Response
