Our study provides evidence that after DSB-generating insults, a mammalian nucleus undergoes a complex compartmentalization reflected by distinct patterns of protein redistribution. The essence of our results is summarized in . For the sake of clarity, the key implications of how the residence sites of the studied proteins help us better understand their roles in the DSB response were systematically discussed while describing the individual spatial categories in the preceding sections. We would like to complement these specific conclusions with more general and conceptual ramifications of the reported results.
In particular, we would like to emphasize that among the diverse modes of protein redistribution after a DSB-generating insult, only the proteins assembled in the DSB-flanking chromatin regions and the ssDNA microcompartments could be readily detected as intranuclear foci. An important implication of this finding is that within the range of physiologically relevant doses of DNA damage, the IRIF formation (and/or the protein assembly at the laser-damaged nuclear tracks) cannot serve as the only criterion for the direct involvement of a given protein in the DSB response. This conclusion is supported by adding several new members to the expanding family of proteins that, although functionally distinct, share the capability of a productive interaction with DSBs without a massive increase in their local concentration. Thus, in addition to signaling kinases whose interaction with the DSB sites is too transient to manifest as foci (Chk1 and -2), other proteins in this category assemble at DSB intermediates whose size is below the resolution of light microscopy (DNA-PK/Ku70). In addition, Smc1 is likely just one example of a larger group of proteins that stably interact with chromatin even in undamaged nuclei and yet become engaged in the DSB signaling and/or repair after local modification by enzymes recruited to the sites of DNA damage.
In conclusion, we hope that the results reported here will provide the necessary framework to add more proteins to the emerging “spatial map” of the DSB-induced genome surveillance network. If coordinated in terms of experimental conditions, subclassification of proteins according to their residence sites before and after DNA damage may help validate, predict, or even exclude their roles in the increasingly complex DSB response. One example illustrating the potential usefulness of the spatial dimension in approaching some lingering questions in the field is the redistribution of the key apical kinases induced by DSBs. Most notably, ATM, ATR, and DNA-PK each occupy distinct nuclear subcompartments (DSB-flanking chromatin, ssDNA microcompartments, and unprocessed DSB ends, respectively). Because deficiency of the respective kinases is accompanied by distinct phenotypes (Abraham, 2004
), it is clear that these kinases, despite sharing several downstream substrates, have limited capability to substitute each other. Although the regulatory network determining the exact function of each of these kinases is very complex, their relocation to distinct compartments after DNA damage can, at least partly, explain their overlapping versus nonoverlapping potential.