FANCF Co-Localizes with Nonerythroid Alpha Spectrin in Nuclear Foci and Shows Enhanced Binding to It in Normal but Not Fanconi Anemia, Group A, Cells after Damage with a DNA Interstrand Cross-Linking Agent.

Abstract

Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, a predisposition to cancer, congenital abnormalities and a cellular hypersensitivity to DNA interstrand cross-linking agents, which correlates with a defect in ability to repair interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in damage-induced nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. The present study was undertaken in order to get a better understanding of the relationship between aSpII and the FA proteins and the functional importance of this relationship in the repair of DNA interstrand cross-links and the repair defect in FA cells. Immunofluorescence microscopy was carried out to determine whether, after damage, additional FA proteins co-localize with aSpII in nuclear foci and whether the interaction between these proteins is enhanced after cross-link damage. The results show that in normal human cells another FA core complex protein, FANCF, co-localizes with aSpII in nuclear foci after cells are damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP). Time course measurements show that these FANCF/aSpII foci are first visible between 6–8 hours after damage and the number of these foci peaks at 16 hours. By 24 hours after exposure, foci are no longer observed. This is the same time frame previously observed for formation and co-localization of FANCA and XPF foci with aSpII. In contrast, in FA-A cells, which are not deficient in FANCF, very few damage induced FANCF or aSpII foci are observed. In corrected FA-A cells, expressing the FANCA cDNA, FANCF and aSpII again co-localize in discrete foci in the nucleus after damage. Co-localization of FANCF in damage-induced foci with aSpII correlates with enhanced binding of FANCF to aSpII after damage. Co-immunoprecipitation studies show that after normal cells are damaged with 8-MOP there is enhanced binding of FANCF, as well as FANCA, to aSpII in the damaged cells compared to this binding in undamaged cells. This further indicates that there is an important interaction between FANCF, FANCA and aSpII during the repair process. These results support our model that aSpII plays a pivotal role in the recruitment of FA and DNA repair proteins to sites of damage where it acts as a scaffold aiding in their interactions with each other or with damaged DNA, thus enhancing the DNA repair process. In FA cells, where there is a deficiency in aSpII, this recruitment is defective as are the interactions of proteins at these sites. This correlates with the reduced repair of interstrand cross-links in FA cells. Thus a deficiency in the interaction of these FA proteins with aSpII may be an important factor in the defective DNA repair pathway in FA cells.