Abstract 2380: Separate but important roles of αSpII and FANCD2 in the FA pathway after DNA interstrand crosslink damage

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, an increased incidence of cancer, genomic instability, congenital abnormalities and a defect in ability to repair DNA interstrand crosslinks (ICLs). We have previously shown that FA cells have a deficiency in the structural protein, nonerythroid alpha spectrin (SpII), which is critical for repair of DNA ICLs and binds to crosslinked DNA. The FA protein, FANCD2, after monoubiquitination (FANCD2-Ub), has also been shown to be critical for ICL repair. However, the relationship between SpII and FANCD2 and whether they are involved in the same steps or events in this process is not known. The present study was undertaken to address these questions and to examine the role of SpII in the function of non-ubiquitinated FANCD2 (non-Ub FANCD2) and FANCD2-Ub in the FA pathway after ICL damage. Immunofluorescence microscopy was used to determine whether these proteins co-localize in nuclear foci after cells are damaged with an ICL agent, 8-methylpsoralen plus UVA light or mitomycin C. Time course measurements showed that formation of FANCD2 foci in normal human cells is different from those of SpII, FANCA and the ICL repair protein, XPF. FANCD2 foci were visible 2 hours after damage, plateaued at 16 hours and were still present at 72 hours. In contrast, SpII, FANCA and XPF foci were visible 10 hours after damage, peaked at 16 hours and by 24 hours were no longer observed. SpII foci, over this time course, did not co-localize with FANCD2 foci after ICL damage and this is corroborated by co-immunoprecipitation studies which show that non-Ub FANCD2 and FANCD2-Ub dissociate from SpII after ICL damage. Studies knocking down expression of SpII by siRNA show that SpII is not needed for monoubiquitination of FANCD2 or its localization to chromatin and nuclear foci after ICL damage. This indicates that SpII and FANCD2-Ub are involved in different steps in the repair process, functioning sequentially, independently and/or in parallel to each other within the FA network. However, in FA-A cells, where FANCD2 is present but is not monoubiquitinated and does not form nuclear foci after ICL damage, restoration of SpII levels to normal by knocking down µ-calpain, a protease which cleaves SpII, leads to formation of FANCD2 foci. However, FANCD2 is not monoubiquitinated. This indicates that SpII is needed for formation of non-Ub FANCD2 nuclear foci after ICL damage and suggests that this may be an important process in the ICL repair response, particularly since we have previously shown that restoration of SpII levels in FA-A cells to normal leads to reversal of a number of the phenotypic deficiencies in these cells. Thus both FANCD2-Ub and non-Ub FANCD2 may be involved in distinct but important steps in the repair process; SpII appears to be of critical importance in the role of non-Ub FANCD2 in the FA pathway and in maintenance of genomic stability after ICL damage. Citation Format: Muriel W. Lambert, Deepa Sridharan, Pan Zhang. Separate but important roles of αSpII and FANCD2 in the FA pathway after DNA interstrand crosslink damage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2380. doi:10.1158/1538-7445.AM2014-2380