These findings suggest an overall intranuclear stabilization of hXPCGFP triggered by binding to lesions.When corrected for the nonresponding cell fraction, UDS levels were more than twofold enhanced.<a href="https://www.ncbi.nlm.nih.gov/pubmed/12130727"></a>
Enhanced repair by increased levels of XPC was confirmed by microinjection of XPCGFP cDNA into homopolykaryons of wildtype human fibroblasts.Interestingly, when a cocktail of XPCGFP and hHRB cDNA was injected, UDS in the majority of the cells was significantly lower and injection of this cocktail appeared highly toxic. These data indicate that large amounts of stabilized XPC may reduce cell viability and suggest that the XPC level is under a delicate control.Because single mutants failed to exhibit any detectable NER defect, the most logical interpretation is that for NER, these proteins are functionally redundant despite their amino acid sequence divergence and size difference. The ability of each of the HR genes to rescue this defect unequivocally established their involvement and fully overlapping function in NER in vivo.DKO cells carried a selective impairment of the GGNER subpathway and apparently normal TCNER, as deduced from the fact that recovery of RNA synthesis after UV irradiation is unaffected.This unresolved mammalianyeast difference is also registered for XPC and its yeast counterpart RAD, underlining the parallels between HR and XPC.The virtual absence of XPC in immunoblots and immunofluorescence directly explained the HR phenocopy of XPC: in the absence of HR, the XPC protein is apparently unstable.This suggests that the steadystate levels of this type of intermediate are very low and that XPC targeted for degradation is rapidly processed.The results derived from the CBZLLL experiments are also relevant in relation to controversies on the functional implications of the RADNERSS proteasome connection.In the absence of proteasome function, yeast conditional S proteasome mutants were reported to display enhanced repair, suggesting that proteins involved in. Consistent with this idea, evidence was reported for RAD ubiquitination and degradation via the S proteasome, involving RAD, although the specific function in DNA repair remained unresolved.On the other hand, overexpression of tagged wildtype RAD caused stabilization of artificial substrates in vivo by preventing multiubiquitination. Physiological levels of RAD were speculated to facilitate substrate degradation, but might also transiently stabilize showed that the S regulatory complex represses NER independently of RAD and apparently independently of proteolysis.Finally, interactions between HRB and the S proteasome were also observed in human cells. Our studies of the HRAB double mutant clearly demonstrate that XPC is intrinsically unstable and is degraded by the S proteasome and that HR proteins partly stabilize XPC in vivo.Consistent with this finding, we were able to alleviate the NER defect caused by HR deficiency, by forced overexpression of exogenous hXPC compensating for the short halflife of endogenous XPC in the absence of HR.The severity of the NER defect in DKO cells correlating with the low cellular XPC content suggests that XPC stabilization is the main in vivo NER function of HR proteins.This phenomenon clearly depends on lesions that are substrates for GGNER and is not due to a general stressrelated response or to any type of DNA damage.XPCGFP induction occurred with grossly similar kinetics when proteasomemediated proteolysis is inhibited, providing further evidence for the idea that protein stabilization is responsible for the XPCGFP increase.