Abstract
Nijmegen breakage syndrome is characterized by genomic instability and a predisposition for lymphoma and solid tumors. Nijmegen breakage syndrome 1 (NBS1), the protein which is mutated in these patients, functions in association with BRCA1 and ATR as part of the cellular response to DNA double-strand breaks. We show here that NBS1 forms foci at the centrosomes via an interaction with gamma-tubulin. Down-regulation of NBS1 by small interfering RNA induces supernumerary centrosomes, and this was confirmed with experiments using Nbs1 knockout mouse cells; the introduction of wild-type NBS1 (wt-NBS1) cDNA into these knockout mouse cells reduced the number of supernumerary centrosomes to normal levels. This phenotype in NBS1-deficient cells is caused by both centrosome duplication and impaired separation of centrioles, which have been observed in BRCA1-inhibited cells. In fact, supernumerary centrosomes were observed in Brca1 knockout mouse cells, and the frequency was not affected by NBS1 down-regulation, suggesting that NBS1 maintains centrosomes via a common pathway with BRCA1. This is consistent with findings that NBS1 physically interacts with BRCA1 at the centrosomes and is required for BRCA1-mediated ubiquitination of gamma-tubulin. Moreover, the ubiquitination of gamma-tubulin is compromised by either ATR depletion or an NBS1 mutation in the ATR interacting (FHA) domain, which is essential for ATR activation. These results suggest that, although centrosomes lack DNA, the NBS1/ATR/BRCA1 repair machinery affects centrosome behavior, and this might be a crucial role in the prevention of malignances.
Highlights
In contrast to normal cells, which have a defined and constant number of chromosomes, the presence of an abnormal number of chromosomes, a condition called aneuploidy, is consistently observed in virtually all human cancers [1, 2]
When the peak sucrose gradient centrifugation fraction containing Nijmegen breakage syndrome 1 (NBS1) was analyzed with immunoprecipitation, NBS1 was coimmunoprecipitated with g-tubulin, MRE11, and BRCA1, indicating a possible physical interaction of the NBS1 complex and g-tubulin in the centrosomes (Fig. 1D)
DNA replication is strictly regulated to prevent the rereplication of chromosomes through a control mechanism involving licensing or tagging, NBS1 down-regulation enhanced the rereplication of chromosomal DNA in the presence of SV40 large T antigen or by overexpression of the licensing factor Cdt1
Summary
In contrast to normal cells, which have a defined and constant number of chromosomes, the presence of an abnormal number of chromosomes, a condition called aneuploidy, is consistently observed in virtually all human cancers [1, 2]. A growing body of evidence suggests that aneuploidy is often caused by chromosomal instability during mitosis [3, 4], which may result from an improper duplication of centrosomes. The occurrence of excess or supernumerary centrosomes is associated. Doi:10.1158/0008-5472.CAN-08-3016 with problems in cell cycle regulation, because centrosomes can continue to duplicate when DNA replication is halted, for example, to repair damage [5,6,7,8]. The disruption of BRCA1 induces supernumerary centrosomes in the absence or presence of hydroxyurea, BRCA1-deficient cells continue DNA replication in the presence of DNA damage [11]. BRCA1 is directly involved in the maintenance of centrosome duplication through the ubiquitination of g-tubulin, the main component of centrosomes, and the disruption of ubiquitination sites results in an excess number of centrosomes [12,13,14]. Supernumerary centrosomes have been frequently observed in the early stages of breast cancer [15, 16]
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