Abstract
The proteins responsible for radiation sensitive disorders, NBS1, kinase ataxia-telangiectasia-(A-T)-mutated (ATM) and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double-strand breaks (DSBs) and are all implicated in checkpoint regulation and DSB repair, such as homologous recombination (HR). We measured the ability of several NBS1 mutant clones and A-T cells to regulate HR repair using the DR-GFP or SCneo systems. ATM deficiency did not reduce the HR repair frequency of an induced DSB, and it was confirmed by findings that HR frequencies are only slightly affected by deletion of ATM-binding site at the extreme C-terminus of NBS1. In contrast, The HR-regulating ability is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1 and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation. Furthermore, normal HR using sister chromatid was completely inhibited by the absence of FHA/BRCT domains. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM-independent fashion.
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Acknowledgements
We are grateful to Dr M Lavin at University of Queensland, Australia and Dr LN Kapp at the University of California, San Francisco, for critical reading of the paper; Dr M Jasin at the Sloan Kettering Cancer Center for the DR-GFP and SCneo constructs; Dr Y Shiloh at Tel Aviv University for ATM cDNAs. We also thank Y Hayuka for her assistance in this work. This work was supported in part by a Grant-in-aid (Kaken-hi) from the Ministry of Education, Science, Sports and Culture of Japan (KK, HT, SS).
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).
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Sakamoto, S., Iijima, K., Mochizuki, D. et al. Homologous recombination repair is regulated by domains at the N- and C-terminus of NBS1 and is dissociated with ATM functions. Oncogene 26, 6002–6009 (2007). https://doi.org/10.1038/sj.onc.1210428
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DOI: https://doi.org/10.1038/sj.onc.1210428
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