Bjorn Rydberg, Sophia Chernikova, Janice Pluth, David J. Chen
and Priscilla K. Cooper
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
CA 94720
DSB rejoining in synchronized populations of repair-defective mutant cell lines. Mutant human and rodent cell lines with defects in genes presumed to affect either non-homologous end-joining (NHEJ) or homologous recombination (HR) processes were tested to determine the relative contribution of each of the pathways to total rejoining of double-strand breaks (DSBs) induced by ionizing radiation (IR). In unsynchronized cells total DSB repair was measured using the standard FAR assay. It was found that NHEJ defective cells show higher levels of unrepaired DSB damage than HR-defective cell lines over the entire 24 hour time period studied. This indicates that in unsynchronized differentiated mammalian cells NHEJ is the primary pathway for repair of IR-induced DSBs, in agreement with results of others. However, even though in unsynchronized cells NHEJ plays the major role, it is possible that in G2 phase of the cell cycle, when both sister chromatids are present, HR may play a larger role. To test whether the contribution to DSB repair by each of the pathways varies in different stages of the cell cycle, we are presently using centrifugal elutriation to separate cells based on size into different cell cycle fractions. As an example of work in progress we will show results for an HR-defective cell line Irs1SF (XRCC3 -/-). The purest G1 and G2 populations of cells were used in the FAR assay to determine DSB repair capabilities for cells in each of these phases of the cell cycle. In both populations of cells repair kinetics were similar, reaching control levels by 6 h after irradiation. However, the non-irradiated control level in G2 cells was significantly higher than control levels in G1 and unsynchronized cells. The result suggests that there is not a large role for HR in rejoining IR-induced DSBs even in G2 cells. Perhaps it plays a supportive role which is not detected unless NHEJ is eliminated, as shown recently for unsynchronized cells by Pluth et al. The fact that higher levels of damage were observed in G2 in control cells could indicate that faulty recombination occurs during S phase in these HR-defective mutants, perhaps when the replication forks encounter endogenous lesions, thus giving rise to DSBs in G2. This possibility will be further examined using synchronized normal cells as well as by testing additional HR mutant cells to see if a similar or different pattern is observed.
Misrejoining of DSBs in NBS cells. The effect of a mutation in the Nbs1 protein, responsible for Nijmegen Breakage Syndrome, was studied using a transformed fibroblast cell line (INBS) from an NBS patient. A normal control fibroblast transformed in the same way (AG1522) was used for comparison. IR-induced DSBs induced in the NBS cell line by 40 Gy of X-rays were fully rejoined by 18 hours when studied with the FAR assay. However, when misrejoining of incorrect ends in a particular large restriction fragment was measured using a hybridization assay developed in our laboratory, preliminary results indicated that misrejoining is more frequent in the NBS line compared with the control. One possible implication of this result is that the Nbs1/Mre11/Rad50 complex may play a role in maintaining close proximity of DNA ends at a DSB, although there are several other alternative possibilities including a role in signaling or recruitment of rejoining pathways with lower probabilities of error.
XPG interaction with proteins involved in DSB repair An overlap between transcription-coupled repair (TCR) and HR processes is suggested by a number of lines of evidence. To examine this possibility directly, we have used the XPG protein, which is required for TCR of oxidative DNA damage, as a probe in Far Western analyses. By this method we have obtained evidence for direct protein-protein interactions of XPG with several proteins required for DNA double-strand break repair, including MRE11, Rad50, the Ku70 subunit of DNA-PK, Rad51, Rad52, and DNA-PKcs. Ku80 displays a consistent, although much weaker interaction, and there is no interaction with XRCC4, which participates in late steps of NHEJ. XPG interactions with Rad51 and Ku70/80 have been confirmed in co-immunoprecipitation experiments. The implications of these interactions are under investigation.
Phosphorylation of XPG in vivo in response to ionizing radiation. Phosphorylation is an important signaling mechanism in regulating DNA repair pathways, and the central role that XPG plays in multiple repair pathways would suggest that XPG might be modified in response to DNA damage. Indeed, we have found that both XPG immunoprecipitated from human cells and recombinant XPG purified from insect cells are constitutively phosphorylated in vivo. Significantly, in vivo labeling experiments demonstrated that phosphorylation of XPG is increased in response to UV and ionizing radiation. The IR-induced hyperphosphorylation is due to the ATM kinase, since it does not occur in cells from an ataxia telangiectasia (AT) patient. Intriguingly, in these cells there is a surprising decrease in phosphorylation after irradiation, indicating that XPG is dephosphorylated in the cellular response to DNA damage. It is likely that in normal cells the damage-induced increase in phosphorylation due to activation of ATM kinase obscured the non-ATM-dependent dephosphorylation that was also occurring. This finding suggests that complex phosphorylation and dephosphorylation schemes are set off in response to IR and that XPG exists in multiple phosphorylation states. In concept, this multi-tiered change in XPG phosphorylation state could regulate the several repair functions of XPG.
Work supported by the Low Dose Program, Office of Biological and Environmental Research, Office of Energy Research, U. S. Department of Energy, under Contract No. DE-AC03-76SF00098; Priscilla K. Cooper (PI).