Abstract 621: XPF, a DNA interstrand crosslink (ICL) repair protein, localizes to telomeres after DNA ICL damage in normal but not Fanconi anemia cells.

Chromosome instability is a hallmark of the genetic disorder, Fanconi anemia (FA), characterized by bone marrow failure and increased cancer susceptibility. The underlying basis for the chromosome instability is not clear, but a defect in repair of DNA ICLs is hypothesized to be a primary cause. The chromosome instability, in particular after ICL formation, suggests telomere dysfunction. We have shown that the structural protein, nonerythroid spectrin (αIISp), is present in the nucleus of normal human cells where it is critical in repair of DNA ICLs and maintenance of chromosome stability and that in FA cells levels of αIISp are reduced due to its increased cleavage by a protease, μ-calpain. Whether chromosomal instability is also linked to defective repair of ICLs in telomeric DNA and to telomere dysfunction is an important question addressed in the present investigation. Studies were specifically carried out to determine whether XPF, a protein involved in repair of DNA ICLs, localizes to telomeres after ICL damage and, if so, whether there is a defect in this association in FA cells and whether this defect can be corrected when levels of αIISp are returned to normal. Normal and FA complementation group A (FA-A) lymphoblastoid cells were treated with a DNA ICL agent, mitomycin C (MMC), and analyzed using immunofluorescence and in situ hybridization (IF-FISH). Cells were probed with a telomeric PNA probe and antibodies against α-spectrin, XPF or two telomeric proteins, TRFI or TRF2. The results show that in normal cells XPF localizes to damage-induced foci at telomeres after ICL damage. In these cells, XPF is present in a diffuse pattern in the nucleus, however, after treatment with MMC a subset of XPF signals colocalize with the PNA probe as well as with TRF1 and TRF2 at telomeres. αIISp was needed for this colocalization. In cells in which αIISp had been knocked down by siRNA and which were treated with MMC, XPF was present in the nucleus but did not colocalize with TRF1 at telomeres. This correlates with the telomere dysfunction observed, characterized by the presence of telomere dysfunction-induced foci (TIF), dramatic loss of telomeres, and production of sister chromatid end-to-end fusions. In FA-A cells, XPF was also present in the nucleus, but it did not localize to telomeres after MMC damage; this was accompanied by significant loss of telomere function. However, when levels of αIISp were restored to normal in FA-A cells by knockdown of μ-calpain, XPF foci colocalized with TRF2 at telomeres in MMC treated cells. These results correlate with increased repair of ICLs and restoration of telomere function and chromosome stability in these cells. These studies indicate that localization of XPF to telomeres after ICL damage is important for ICL repair and telomere function and that a defect in repair of ICLs in telomeres in FA cells may be an important factor in the chromosome stability observed. Citation Format: Pan Zhang, Muriel W. Lambert. XPF, a DNA interstrand crosslink (ICL) repair protein, localizes to telomeres after DNA ICL damage in normal but not Fanconi anemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 621. doi:10.1158/1538-7445.AM2013-621