Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines

Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA poly merase delta and epsilon, is involved in both DNA replication and repair. P revious studies in vitro have demonstrated the requirement of PCNA in the r esynthesis step of nucleotide excision repair (NER) and base excision repai r (BER). Using a native chromatin template isolated under near physiologica l conditions, we have analysed the involvement of PCNA in the BER pathway i n different NER defective human cell lines. The repair sites and PCNA were visualized by indirect immunolabelling followed by fluorescence microscopy, The results indicate that exposure to X-rays triggers the induction of PCN A in all the three human fibroblast cell lines studied, namely normal, xero derma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B), In a ll the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient X P-A cells suggests that the X-ray-induced lesions are largely repaired via the BER pathway involving PCNA as one of the key components of this pathway . X-ray-induced repair synthesis was greatly inhibited by treatment of cell s with DNA polymerase inhibitors aphidicolin and cytosine arabinoside, Inte restingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be requir ed for the BER pathway at a step preceding the resynthesis step.