The response of proteasome activator 200kD to DNA damage.

摘要

Upon exposure to DNA damaging agents, eukaryotic cells employ a complex cellular response, which includes cell signaling and protein recruitment events, in order to sense and repair DNA damage. The correction of DNA damage-induced lesions is fundamental for both cell survival and the maintenance of genomic stability. Proteasomes are the chief protein degradation machinery within the cytoplasm and nucleus of the cell and several components of proteasomes have been implicated in the cellular response to DNA damage. These components include 20S catalytic core proteasomes, ubiquitin-binding 19S regulatory caps and PA200; the latter is known to enhance 20S proteasome mediated cleavage after acidic residues (post-glutamyl activity) most dramatically. In our assessment of the interactions between these proteasome components; we find that most, if not all, cellular PA200 binds specifically to 20S core proteasomes. Furthermore, we find that PA200-20S proteasomes can exist as a hybrid with the 19S regulatory complex (PA200-20S-19S) and that the abundance of these PA200-19S hybrid proteasomes is increased by exposure to ionizing radiation (IR), which induces DNA breaks. Exposure of cells to IR induced the accumulation of subunits of PA200-19S hybrid proteasomes, as well as proteolytic activity on chromatin. Chromatin immunoprecipitation (ChIP) assays in yeast cells demonstrated that this accumulation is likely at the site of DNA damage as yeast PA200 associated specifically with DNA strand breaks. Unlike other DNA damage responding proteins (NBS1 and RAD51), enhanced levels of PA200-19S hybrid proteasomes on chromatin was detected at later time points following IR exposure and this accumulation appeared independent of both the DNA repair kinase ATM and the DNA repair mediator p53. These critical responders to DNA damage are required for the recruitment and accumulation of many other repair proteins on chromatin, which suggests a unique mode of recruitment for PA200. Regardless of the differences in recruitment between PA200-19S proteasomes and known DNA damage responders, the importance of PA200 is similar to many proteins that respond to DNA damage in that PA200-knockdown cells exhibit reduced survival after exposure to IR as well as genomic instability, even in the absence of exogenous DNA insults. This phenotype is reproduced by specific inhibition of post-glutamyl proteasome activity suggesting an important role for PA200 in genomic stability through post-glutamyl cleavage events. This work provides the first direct evidence that PA200 is important for the cellular response to IR and in maintaining genomic stability.