This along with existing literature that yeast 20S and 19S proteasome subunits are recruited to sites of DNA repair supports the idea that degradation machinery may be a crucial step of the response (35)

This along with existing literature that yeast 20S and 19S proteasome subunits are recruited to sites of DNA repair supports the idea that degradation machinery may be a crucial step of the response (35). or bleomycin (19). Second, mice lacking PA200 show normal lymphocyte development, NVP-BEP800 a process that requires DNA repair proteins to rearrange antigen receptor gene NVP-BEP800 segments (21). Based on NVP-BEP800 these observations, the importance of PA200 in response to DNA damage and in DNA repair have been questioned. However, it is conceivable that PA200 plays an important role in response to IR-induced damage but that this role occurs after DNA repair is complete, for example. Consistent with the notion that PA200 can impact the cellular response to DNA damage, Schmidt (19) observed bleomycin sensitivity of yeast expressing a deletion TM4SF2 mutant lacking the C terminus of Blm10. In the current work, we characterized how PA200 responds to IR NVP-BEP800 and determined the importance of PA200 in survival from IR-induced damage. We provide evidence that NVP-BEP800 PA200-19S hybrid proteasomes are enhanced after IR exposure and accumulate on chromatin. Furthermore, PA200 and postglutamyl proteolytic activity are required for the cellular response to IR and maintenance of genomic integrity. Results Elevated Levels of PA200-19S Hybrid Proteasomes After IR Exposure. To demonstrate that PA200 and 19S caps could interact with either end of the same core proteasome to form hybrid PA200-20S-19S proteasomes (henceforth referred to as PA200-19S hybrid proteasomes) we immunoprecipitated 19S cap subcomplexes and show associated PA200 and core proteasomes (Fig. 1(23, 24)]. This pNBS1 is maintained until at least 6 h after IR exposure but disappears by 24 h (Fig. 2and were assessed for proteasome activity 24 h after IR exposure (*, 0.001). Specific fluorescence was calculated by subtraction of the fluorescence released in the presence of inhibitor from the absence of inhibitor. Error bars are SD of triplicates. (and (4), is enhanced 19-fold on chromatin (Fig. 2and and supporting information (SI) Fig. S1 and and were quantified as fluorescence normalized to KU70 protein levels. (and and Fig. S1 and and Fig. S1 0.05) compared with control siRNA cells or untransfected cells (Fig. 4 0.01) decrease in cell survival after IR exposure (Fig. 4= 0.05). Cells treated with a combination of both PA200 siRNA and YU-102 show survival defects comparable with YU-102 treatment alone (Fig. 4 0.05, by 2 analysis). ( 0.01, by 2 analysis; *, 0.05). Error bars indicate SD from three independent experiments. PA200 and Postglutamyl Proteasome Activity Contribute to Genomic Stability. Because sensitivity to IR, as measured by decreased cell survival after IR exposure, is often correlated with genomic instability (30), we speculated that PA200 deficiency and diminished postglutamyl proteasome activity may result in genomic instability. In support of this idea, the number of chromosome aberrations (end-to-end associations, breaks and gaps, bridges) in PA200-depleted cells were significantly greater compared with control treated cells even in the absence of IR exposure (Table 1). Correspondingly, cells treated with YU-102 showed dramatically ( 0.01) increased genomic instability as measured by micronuclei formation (Table 2). Because both PA200 knockdown and inhibition of postglutamyl proteasome activity led to genetic instability and decreased survival after IR, these findings further support the notion that PA200 functions together with core proteasomes during the cellular response to IR-induced DNA damage. Table 1. Chromosome abnormalities on PA200 siRNA 0.05. Table 2. Proteasome inhibitors induce genetic instability 0.01; *, = 0.05. Discussion Overall, we have presented several lines of evidence indicating that PA200 responds to DNA damage in a manner that is essential for cell survival and that PA200 is critical to maintain genomic stability. Our analysis shows that PA200 responds to IR by increasing levels of PA200-20S-19S hybrid proteasomes and that these complexes.