Abstract 3018: FANCF, a Fanconi anemia core protein, functions outside of monoubiquitinating FANCD2 in DNA interstrand crosslink repair

Abstract

Abstract Genomic instability is a hallmark of the genetic disorder, Fanconi anemia (FA), which is characterized by bone marrow failure, an increased incidence of cancer, congenital abnormalities and a defect in ability to repair DNA interstrand cross-links (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid A spectrin (ASpII), which is critical for repair of DNA ICLs and binds to cross-linked DNA. Eight FA proteins, FANC -A, B, C, E, F, G, L, and M, form a core complex which is essential for monoubiquitination of FANCD2, an important step in ICL repair. However, whether any of these core proteins play additional roles in the ICL repair process 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 the ICL repair process with ASpII. Immunofluorescence microscopy was used to determine whether these two proteins co-localize in nuclear foci after normal human cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light (8-MOP). Time course measurements showed that, in normal human cells, FANCF nuclear foci formed over a similar time course as did those of ASpII. This time course was also similar to that of FANCA and the ICL repair protein, XPF. FANCF foci, as well as ASpII, FANCA and XPF foci, were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. FANCF foci, over this time course, co-localized with ASpII foci, indicating that FANCF is associated with ASpII during ICL repair. This association was corroborated by co-immunoprecipitation studies which demonstrated that FANCF has enhanced binding to ASpII after ICL damage. This indicates that FANCF is involved in the same steps in the repair process as ASpII. 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 colocalized with ASpII foci. Our studies indicate that this is due to restoration of ASpII levels to normal. These studies support a model we have proposed in which ASpII acts as a scaffold in the recruitment of proteins (i.e., FANCF, FANCA, and XPF) to sites of ICL damage and that FA proteins, such as FANCA, are needed for maintenance of ASpII stability in the cell. In the transfected FA-A cells, expression of FANCA leads to enhanced stability of ASpII, which then participates in the ICL repair process. These studies indicate that the FA core complex protein, FANCF, has an additional function in ICL repair, besides monoubiquitination of FANCD2, and is involved with ASpII in its role in the repair process and in maintaining genomic stability after DNA ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. FANCF, a Fanconi anemia core protein, functions outside of monoubiquitinating FANCD2 in DNA interstrand crosslink repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2015-3018