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. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid spectrin (α SpII∑*) and that this deficiency correlates with a defect in ability to repair DNA interstrand cross-links. In order to get a better understanding of the exact role that α IISp∑* plays in the repair of cross-links and the repair defect in FA, whether it may have additional and potentially critical functions in the nucleus, and the processes that might be most severely affected by a defect in this protein, studies were undertaken to determine precisely what other proteins α IISp∑* interacts with in the nucleus. Co-immunoprecipitation experiments were carried out in which chromatin-associated proteins from normal human lymphoblastoid cells that co-immunoprecipitated (Co-IP) with α II spectrin were examined and identified. These proteins could be grouped into five categories: structural proteins, proteins involved in DNA repair, chromatin remodeling proteins, FA proteins, and transcription and RNA processing proteins. The structural proteins that Co-IP with α II spectrin were: lamin A, actin, protein 4.1B, β IV spectrin, and emerin. This indicates that α II spectrin interacts with proteins in the nucleus that play a role in nuclear cytoskeleton stability, chromatin organization and transcription. A number of proteins that Co-IP with α II spectrin were involved in DNA repair: DNA interstrand cross-link repair (XPF), homologous recombinational repair (HRR) and non-homologous end joining (NHEJ) (MRE11, RAD 50, RAD 51, XRCC2, Ku 70, Ku 80), and nucleotide excision repair (NER) (hHR23B, XPA, RPA, XPB, XPG, XPF, ERCC1). Since both NER and HRR are thought to be involved in repair of DNA interstrand cross-links, association of α II spectrin with XPF and HRR proteins supports our hypothesis that α II spectrin acts as a scaffold for recruitment and alignment of repair proteins at sites of DNA damage. It may act as a scaffolding for proteins involved in more than one repair pathway. α II spectrin also associated with chromatin remodeling proteins: BRG1, hBRM and CSB. This indicates that, like actin, it not only plays a role in nuclear cytoskeletal structure but also in chromatin remodeling as well. In agreement with our previous findings, α II spectrin Co-IP with FANCA and FANCC. The present study showed that it also Co-IP with FANCD2, FANCG and FANCF. There was also a significantly greater association of several FANC proteins, such as FANCA, to α II spectrin after cross-link damage to the cells than in undamaged cells. This further indicates that there is an important interaction between these FANC proteins and α II spectrin during the repair process. Several proteins involved in transcription and RNA processing (p40 and hnRNP A2/B1) also Co-IP with aII spectrin. Again, like actin, aII spectrin in the nucleus may also be involved in these processes. These results indicate that aII spectrin may have multiple roles in the nucleus and, in addition to DNA repair, may be involved in processes such as nuclear cytoskeleton stability, chromatin remodeling, transcription and RNA processing. A deficiency in aII spectrin in FA cells could thus affect multiple pathways where interaction of aII spectrin with functionally important proteins is critical; loss of this interaction in FA cells may explain some of the diverse clinical characteristics of this disorder.
The Fanconi Anemia Pathway in Cancer
