Kobayashi Junya, Tauchi Hiroshi, Sakamoto Shuichi, Nakamura Asako, Morishima Ken-ichi, Matsuura Shinya, Kobayashi Toshiko, Tamai Katsuyuki, Tanimoto Keiji, Komatsu Kenshi
Department of Oral and Maxillo Facial Radiology, Hiroshima University, Hiroshima 734-8553, Japan.
Curr Biol. 2002 Oct 29;12(21):1846-51. doi: 10.1016/s0960-9822(02)01259-9.
DNA double-strand breaks represent the most potentially serious damage to a genome; hence, many repair proteins are recruited to nuclear damage sites by as yet poorly characterized sensor mechanisms. Here, we show that NBS1, the gene product defective in Nijmegen breakage syndrome (NBS), physically interacts with histone, rather than damaged DNA, by direct binding to gamma-H2AX. We also demonstrate that NBS1 binding can occur in the absence of interaction with hMRE11 or BRCA1. Furthermore, this NBS1 physical interaction was reduced when anti-gamma-H2AX antibody was introduced into normal cells and was also delayed in AT cells, which lack the kinase activity for phosphorylation of H2AX. NBS1 has no DNA binding region but carries a combination of the fork-head associated (FHA) and the BRCA1 C-terminal domains (BRCT). We show that the FHA/BRCT domain of NBS1 is essential for this physical interaction, since NBS1 lacking this domain failed to bind to gamma-H2AX in cells, and a recombinant FHA/BRCT domain alone can bind to recombinant gamma-H2AX. Consequently, the FHA/BRCT domain is likely to have a crucial role for both binding to histone and for relocalization of hMRE11/hRAD50 nuclease complex to the vicinity of DNA damage.
DNA双链断裂对基因组而言是最具潜在严重性的损伤；因此，许多修复蛋白通过目前特征尚不明确的传感机制被招募至核损伤位点。在此，我们表明，尼曼匹克氏症候群（NBS）中存在缺陷的基因产物NBS1通过直接结合γ-H2AX与组蛋白而非受损DNA发生物理相互作用。我们还证明，NBS1的结合可在不与hMRE11或BRCA1相互作用的情况下发生。此外，当将抗γ-H2AX抗体导入正常细胞时，这种NBS1的物理相互作用会减弱，并且在缺乏H2AX磷酸化激酶活性的共济失调毛细血管扩张症（AT）细胞中也会延迟。NBS1没有DNA结合区域，但具有叉头相关（FHA）结构域和BRCA1 C端结构域（BRCT）的组合。我们表明，NBS1的FHA/BRCT结构域对于这种物理相互作用至关重要，因为缺乏该结构域的NBS1在细胞中无法与γ-H2AX结合，并且单独的重组FHA/BRCT结构域就能与重组γ-H2AX结合。因此，FHA/BRCT结构域可能对于与组蛋白的结合以及hMRE11/hRAD50核酸酶复合物重新定位到DNA损伤附近均起着关键作用。
