Abstract
TopBP1, which has been isolated as an interacting protein with DNA topoisomerase II beta, has eight BRCT domains, and shares sequence homology with yeast Cut5/Rad4. It has been reported that TopBP1 forms foci at the sites with DNA damages, which were co-localized with those of gamma-H2AX, NBS1, PCNA, and BRCA1. TopBP1 is phosphorylated by ATM, and is involved in Chk1 and Chk2 phosphorylation after DNA damage. TopBP1 knockdown cells using anti-sense oligonucleotide showed increase sensitivity to DNA damaging agents. Here, we show that TopBP1 physically interacts with NBS1, which is the underlying protein for Nijmegen breakage syndrome (NBS). The formation of TopBP1 foci was significantly inhibited in NBS cells after irradiation, whereas NBS1 foci formation was not affected in the cells with reduced TopBP1 expression by RNAi. Formation of TopBP1 foci was also inhibited in AT cells. Physical interaction between TopBP1 and NBS1 was increased after irradiation, and NBS cells transfected with S343A mutant showed reduced formation of TopBP1 foci. These results suggested that TopBP1 is a downstream protein of NBS1, and the physical interaction with NBS1 requires phosphorylation of NBS1 by ATM.
