Localization of αII Spectrin to Telomeres After DNA Interstrand Cross-Link Damage Is Deficient In Fanconi Anemia Group A Cells, Which May Lead to Telomere Dysfunction.

Abstract

AbstractAbstract 1160A hallmark of the bone marrow failure disorder, Fanconi anemia (FA), is genomic instability which is characterized by both chromosome instability and defects in repair of DNA interstrand cross-links (ICLs). The chromosome instability in FA, in particular chromosome end fusions and breaks, suggests that there is telomere dysfunction. We have shown that the structural protein, nonerythroid a spectrin (aIISp), is present in the nucleus of normal human cells where it is critical in both repair of DNA ICLS and maintaining chromosome stability. aIISp is important in recruitment of the ICL repair protein, XPF, to sites of damage. We have shown that there is a deficiency in aIISp in FA cells lines, due to its increased breakdown, which correlates with reduced repair of DNA ICLs and increased chromosomal aberrations after ICL damage. The goal of the present study was to determine whether aIISp plays a role in telomere function after DNA ICL damage and whether telomere dysfunction in FA cells after damage can be corrected when levels of aIISp are restored to normal. Normal human and FA, complementation group A (FA-A) lymphoblastoid cells, which have telomerase, were used. Undamaged cells or cells treated with mitomycin C (MMC), a DNA ICL agent, were studied using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with anti-a-spectrin and either (C3TA2)3-Cy3 labeled peptide nucleic acid (PNA) telomeric probe or antibodies against two proteins, TRF1 or TRF2, which are components of the shelterin complex that is essential for protecting chromosome ends and preventing telomere dysfunction. The appropriate fluorescent secondary antibodies were used. The results showed that 47–50% of aIISp foci colocalized with the PNA probe, TRF1 or TRF2 in telomeres in MMC damaged but not undamaged normal cells. Levels of colocalization peaked at 16 hours and, by 24 hours, aIISp foci were no longer observed. This colocalization was specific for the S phase of the cell cycle. These results showed that in normal cells aIISp associates with telomeres in S phase after ICL damage and may play an important role in telomere function after damage. After ICLs are presumably unhooked/removed, aIISp dissociates from telomeres. The view that aIISp is involved in telomere function after ICL damage is strengthened by coimmunoprecipitation studies which show that aIISp has markedly enhanced binding for TRF1 and TFR2 after cells were damaged with MMC. IF-FISH also showed that the ICL repair protein, XPF, had enhanced association with telomeres after ICL damage, which further indicated that repair is taking place. In FA-A cells, in which aIISp levels are reduced, aIISp did not colocalize with the PNA probe, with TRF1 or with TRF2 after ICL damage; XPF also did not localize to telomeres after ICL formation. However, after aIISp levels were returned to normal in FA-A cells by siRNA knock down of m-calpain, a protease which cleaves aIISp and levels of which are elevated in FA cells, aIISp as well as XPF associated with telomeres after ICL damage. The chromosomal aberrations (i.e., breaks and end fusions) observed in FA-A cells after ICL damage were also corrected. ICLs can lead to stalled DNA replication forks in S phase. The present results suggest that, after damage, aIISp associates with telomeres in S phase at ICLs at stalled replication forks. Since aIISp is involved in ICL repair, this association could be of particular importance in repair of ICLs in replicating telomeres. In FA-A cells, failure of localization of aIISp and XPF to telomeres after ICL damage could have severe consequences. It could lead to unrepaired ICLs, which in replicating telomeres could lead to telomere dysfunction and to production of the chromosomal aberrations observed in these cells. These studies show that aIISp may play an important role in telomere function after DNA damage and during telomere replication. Correction of the deficiency in aIISp in FA cells by knocking down m-calpain could be significant in reversing telomere dysfunction and chromosomal aberrations after ICL damage and could potentially have important therapeutic relevance. Disclosures:No relevant conflicts of interest to declare.