Functional Interaction between FANCD2 and ATM in the DNA Damage Response.
Authors: Margossian Steven P. , Ho Gary , Kennedy Richard , D’Andrea Alan D. 
Published: April 2020

Abstract
Fanconi Anemia is a multigenic cancer susceptibility syndrome. The FA pathway controls the monoubiquitination of FANCD2, and its targeting to damage-associated DNA foci. Cells that are deficient in the FA pathway are highly sensitive to bi-functional crosslinking agents such as mitomycin-C (MMC). Although FANCD2 monoubiquitination is activated by DNA damage, how this is coupled to the DNA damage response is unknown. Previous studies suggest a functional interaction between FANCD2 and the protein kinase ATM (ataxia telangiectasia mutated). Recently we have shown that the C-terminus of FANCD2 is required for its function (Montes de Oca et al., Blood2005:105, 1003). A C-terminal truncation of FANCD2 (FANCD2-C) fails to correct the MMC sensitivity FANCD2−/− cells despite its monoubiquitination and assembly into DNA damage foci. In the present study we demonstrate that FANCD2 forms a protein complex with the ATM kinase and that this interaction requires the FANCD2 C-terminus. The association of ATM with FANCD2 occurs in the absence of DNA damage, and does not require FANCD2 to be monoubiquitinated. In immunoprecipitation experiments involving the FANCD2-C mutant, ATM fails to co-precipitate, suggesting the C-terminus of FANCD2 is required for recruiting ATM to sites of DNA damage. In addition, we have identified a new ATM phosphorylation site on FANCD2, Thr691. Using a phospho-specific antibody to FANCD2 Thr691 we have determined that siRNA knockdown of ATM results in loss of phosphorylation of Thr691, and ionizing radiation (IR) activates the phosphorylation of Thr691. Mutation of Thr691 to Alanine destroys this ATM phosphorylation site. Interestingly, this FANCD2 mutant also fails to correct the MMC sensitivity FANCD2−/− cells even though it can be monoubiquitinated and targeted to DNA foci following DNA damage, a phenotype observed for the FANCD2-C mutant. Next we examined the phosphorylation of Thr691 in FANCD2-C. In FANCD2 constructs expressing a mutated or truncated C-terminus, phosphorylation of Thr691 is delayed following exposure to MMC and the cells are sensitive to DNA damage. Following exposure to ionizing radiation the ATM kinase phosphorylates itself on Ser1981. Using a phospho-specific antibody to ATM Ser1981 we evaluated the phosphorylation of ATM in response to DNA damage caused by IR and MMC. Western analysis shows that phosphorylation of ATM on Ser1981 following IR is the same in FANCD2 and FANCD2-C cells as well as uncorrected FAND2−/− cells. ATM foci formation following IR also appears identical in all three FANCD2 cell lines based upon immunoflourescence microscopy. However in cells damaged with MMC, there is a delay in Ser1981 phosphorylation and fewer ATM foci in the uncorrected and FANCD2-C cells compared to cells corrected with intact FANCD2. We propose a model for FANCD2 function where the targeting of FANCD2 to DNA damage inducible foci is required for the proper coordination of some ATM-dependent DNA repair and checkpoint responses. For instance, FANCD2 may be required for targeting ATM to a subset of double strand breaks - namely, those breaks associated with an adjacent interstrand crosslink. Thus targeting of ATM to MMC-induced DNA damage requires FANCD2 but recruitment to IR-induced double strand breaks does not, depending instead on NBS1 and the M/R/N complex.