Multiple Roles of NBS1 for Genotoxic and Nongenotoxic Stresses

Abstract

Nijmegen breakage syndrome (NBS) is characterized by genome instability and microcephaly. We show here that NBS human cells are severely sensitive to mitomycin C, similar to Fanconi anemia (FA). To study the details of interstrand cross-link (ICL) repair in NBS and FA, a highly sensitive psoralen-PEO-biotin (PPB) dot blot assay was developed to provide sensitive quantitative measurements of ICLs during the removal process. Studies utilizing this assay demonstrated a decreased rate of ICL removal in cells belonging to the FA core complex group (e.g., groups A and G), but ICL removal was restored to normal levels after these cells were complemented with wt-FANCA and wt-FANCG. In addition, a defect in ICL removal was also observed in cells with a mutated FANCD2 protein. Conversely, FA-D1 cells with a defective BRCA2 protein display normal ICL removal, although they are compromised with respect to recombination. We also show that cells from patients with NBS were defective in ICL removal, whereas they are impaired in recombination, similar to that of BRCA/XRCC3-defective cells. On the other hand, NBS1 is implicated in a response to nongenotoxic stress. We show here that the subpopulation of NBS I is localized at the centrosomes and has a crucial role for maintenance of the centrosomes. Downregulation of NBS I by siRNA induces Supernumerary centrosomes, and this was confirmed by experiments using NBS1 knockout mouse cells. Although introduction of wt-NBS1 cDNA into knockout mouse cells restores the number of centrosomes to normal levels, it is abolished after the introduction of NBS I mutated at the FHA domain and at a serine residue of 343, both of which are essential regions for ataxia-telangiectasia and Rad3-related protein (ATR) activation. The present results indicate the involvement of NBS I in a response to genotoxic stress and in maintenance of centrosome through the ATR pathway.