Nonerythroid nuclear A spectrin (ASpII) is critical for repair of DNA interstrand crosslinks (ICLs) and for genomic stability. We have previously shown that there is a deficiency in ASpII in the inherited chromosomal instability disorder, Fanconi anemia (FA), which has a defect in ability to repair DNA ICLs and a predisposition to cancer. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex essential for monoubiquitination of FANCD2 (FANCD2-Ub), a process which is critical for ICL repair. However, whether any of these FA core proteins play additional roles in ICL repair is not clearly known. The present study was undertaken to address this question and to examine whether one of these proteins, FANCF, is involved in steps in the ICL repair process in which ASpII also plays a role. Immunofluorescence microscopy was used to determine whether FANCF co-localizes with ASpII in nuclear foci in normal human cells after they are damaged with an ICL agent, 8-methoxypsoralen plus UVA light (8-MOP). Time course measurements showed that FANCF co-localized in nuclear foci with ASpII and followed a similar time course for formation. This time course was similar to that of the ICL repair protein, XPF, which produces incisions at sites of ICLs and acts downstream of FANCD2-Ub. FANCF foci, like ASpII and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. This association of FANCF with ASpII was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. These studies indicate that FANCF associates with ASpII and is involved with ASpII in the repair process. Since we have demonstrated that ASpII, like XPF, acts downstream of FANCD2-Ub and that FANCF co-localizes with ASpII after ICL damage, this suggests that FANCF interacts with ASpII in repair events downstream of FANCD2-Ub. In FA-A cells, FANCF is present as in normal cells, but does not form nuclear foci after ICL damage. Transfection of FA-A cells with a cDNA expressing FANCA, however, led to restoration of ASpII levels to normal and to formation of FANCF nuclear foci, which co-localized with ASpII foci. This indicates that ASpII is needed in localization of FANCF to sites of damage. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins involved in the repair process to sites of ICL damage. They also show that FANCF has an additional function in ICL repair besides monoubiquitination of FANCD2. We propose that after DNA damage and monoubiquitination of FANCD2, ASpII and FANCF act downstream of FANCD2, along with XPF, and that the interaction between these proteins at sites of damage is critical for the repair process and maintenance of genomic stability.
Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core complex protein involved in monoubiquitination of FANCD2, also has a role with nuclear alpha spectrin in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2758.