The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites was investigated in human quiescent fibroblasts after UV irradiation. Associated PCNA was detected with the monoclonal antibody (MoAb) PC10 and by immunofluorescence assessment with flow cytometry, after a hypotonic lysis step in order to release unbound molecules. Immunofluorescence levels, relatively low in untreated control cells, increased by about threefold after uv irradiation. The time course of PCNA complex formation showed a maximum after about 30 min from irradiation and was found to be dose-dependent up to about 10 J/m2, after that it reached a plateau. Formation of the PCNA-chromatin complex was neither significantly affected by the topoisomerase II inhibitor VP-16, nor by the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide. In contrast, a significant reduction was obtained either after ATP depletion or after incubation with the protein-kinase inhibitor staurosporine. Immunoprecipitation studies on nuclear extracts from 32 P-labeled cells showed that PCNA bound to DNA synthesis sites was phosphorylated. The results indicate that PCNA associated to DNA repair synthesis sites may be detected with PC10 MoAb after a hypotonic lysis step, and provide evidence that the transition from soluble to chromatin-associated form of the protein is dependent on a phosphorylation mechanism.
Proliferating cells nuclear antigen complex formation induced by ultraviolet irradiation in human quiescent fibroblasts as detected by immunostaining and flow cytometry
STIVALA, LUCIA ANNA;BIANCHI, LIVIA
1993-01-01
Abstract
The association of the proliferating cell nuclear antigen (PCNA) to DNA synthesis sites was investigated in human quiescent fibroblasts after UV irradiation. Associated PCNA was detected with the monoclonal antibody (MoAb) PC10 and by immunofluorescence assessment with flow cytometry, after a hypotonic lysis step in order to release unbound molecules. Immunofluorescence levels, relatively low in untreated control cells, increased by about threefold after uv irradiation. The time course of PCNA complex formation showed a maximum after about 30 min from irradiation and was found to be dose-dependent up to about 10 J/m2, after that it reached a plateau. Formation of the PCNA-chromatin complex was neither significantly affected by the topoisomerase II inhibitor VP-16, nor by the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide. In contrast, a significant reduction was obtained either after ATP depletion or after incubation with the protein-kinase inhibitor staurosporine. Immunoprecipitation studies on nuclear extracts from 32 P-labeled cells showed that PCNA bound to DNA synthesis sites was phosphorylated. The results indicate that PCNA associated to DNA repair synthesis sites may be detected with PC10 MoAb after a hypotonic lysis step, and provide evidence that the transition from soluble to chromatin-associated form of the protein is dependent on a phosphorylation mechanism.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
