53BP1, a member of the BRCT protein family, is hyperphosphorylated and relocalizes to a number of nuclear foci in response to DNA damage. These foci also contain the Mre11-Rad50-Nbs1 complex. 53BP1, however, concentrates in foci in 30 min, which is significantly earlier than the time any obvious concentration of Mre11 occurs in the postirradiation period (9 to 15 h after irradiation [33
]). There were several proteins that coimmunoprecipitated with 53BP1 (Fig. A). However, we detected neither p53 nor Mre11 in the 53BP1 immunoprecipitate under the experimental conditions (data not shown).
The assembly of 53BP1 foci is not cell cycle stage dependent. In contrast, Rad51 and BRCA1, which also change their subnuclear localization after DNA damage, show a focal distribution in S phase and disperse homogeneously over the nucleoplasm after genotoxic treatments (52
). Ionizing irradiation induces Rad51 and BRCA1 focal assembly in cells presumed to be in either G1 or G2 (15
), whereas irradiation-induced 53BP1 foci occur in almost all cells except those in mitosis. Rad51, Mre11, or BRCA1 foci are observed in a subset of cells after irradiation (10 to 40%, depending on the protein, cell type, and cell cycle stage [33
The histone H2A variant H2AX, which makes up 20% of the total H2A in human cells, is phosphorylated at Ser 139 in response to dsDNA breaks. H2AX phosphorylation has been demonstrated to occur at the sites of lesions by scissoring laser treatment (46
). Recently, a comprehensive study has shown that the different types of damage-induced foci, including BRCA1, Rad51, and the Mre11-Rad50-Nbs1 complex foci, all originate from γ-H2AX foci at certain stages of the postirradiation period (44
). The associations appear to follow a sequential order, and some of them might also be cell cycle stage dependent. The focally distributed H2AX phosphorylated at Ser 139 (called γ-H2AX) appears immediately after irradiation, peaks by 10 min, and then gradually decreases and almost disappears after 4.5 h (46
). Kinetically, the appearance of 53BP1 foci and 53BP1's hyperphosphorylation occur slightly later than the appearance of foci and H2AX's hyperphosphorylation of H2AX but earlier than with the other types of damage-induced nuclear foci. At 30 min postirradiation the focal distribution and the phosphorylation of XL53BP1 are not at their maxima (Fig. and ). This result might be reasonable since 53BP1 needs to be recruited to the foci after DNA damage and, in contrast, since H2AX is already there at the dsDNA breaks. More than half of the dsDNA breaks are rejoined in an order of minutes, and the rest persist for several hours (26
). 53BP1 foci might be implicated in the repair and/or checkpoint control associated with the latter.
53BP1 is hyperphosphorylated in response to ionizing irradiation, and this phosphorylation is significantly reduced in AT cells which are deficient in ATM kinase (Fig. C). The data obtained with the inhibitors of ATM-related kinases are consistent with the AT cell results (Fig. B). These results suggest that ATM-related kinases are involved in the phosphorylation of 53BP1 in response to ionizing irradiation. Interestingly, caffeine inhibits 53BP1 phosphorylation more efficiently than wortmannin. This is also true for the suppressive effects of caffeine and wortmannin on the relocalization of 53BP1: caffeine blocks and wortmannin slightly delays 53BP1 focus formation (Fig. and Table ). We observed a slight delay in 53BP1 focal assembly in AT cells after irradiation (Fig. and Table ). This suggests that ATM kinase might be involved in the rapid response of 53BP1 relocalization to ionizing irradiation but that it is not essential for formation of the foci. Mre11/Rad50 focus formation is reduced in AT cells but not completely abolished (33
). BRCA1 focus formation is not different in wild-type cells and in AT cells (10
). ATM, ATR, and DNA-PK might have overlapping roles in phosphorylation and in focus formation. During our paper's revision, Xia et al. published a paper showing that ATM kinase phosphorylates XL53BP1 in vitro, which is consistent with our observation of the inhibitors and AT cells (66
Our results suggest that the formation of 53BP1 foci is controlled through phosphorylation of the protein. In budding yeast, accumulating evidence implicates phosphorylation-regulated protein-protein interactions in the cellular response to DNA damage. dsDNA breaks induce relocalization of Ku, Rap1, and SIR proteins, which are normally associated with telomeres, to the sites of the lesion, and this process is dependent on MEC1sc
, an ATM-related kinase, and Rad9sc
, a BRCT protein (32
protein multimerizes through its BRCT domain, and the interaction occurs with higher affinity after DNA damage (56
). The FHA domain of Rad53sc
physically interacts with phosphorylated forms of Rad9sc
). Given the fact that there is no evidence for the presence of an active transport system in the nucleus at the moment and that RNA and protein factors move by diffusion through the interchromatin space (see reference 30
), it might be reasonable to speculate that the assembly and disassembly of repair and checkpoint protein foci are regulated through changing of the affinities of protein-protein interactions by phosphorylation (see also reference 44
The results presented here implicate 53BP1 in DNA damage repair or checkpoint control in vertebrate cells; however, direct evidence is still missing. A genetic approach (i.e., disruption of the 53BP1 gene in DT40 or ES cells) will be required to investigate these possibilities directly.