Mutagenesis, Vol. 16, No. 3, 225-232,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Analysis of repair and PCNA complex formation induced by ionizing radiation in human fibroblast cell lines
MGC, Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, PO Box 9503, 2300 RA Leiden, Wassenaarseweg 72,The Netherlands and 1 Center for Radiological Research, Columbia University, College of Physicians and Surgeons, 630W 168th Street, New York, NY 10032, USA
Proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerase 
and 
, is involved in both DNA replication and repair. Previous studies in vitro have demonstrated the requirement of PCNA in the resynthesis step of nucleotide excision repair (NER) and base excision repair (BER). Using a native chromatin template isolated under near physiological conditions, we have analysed the involvement of PCNA in the BER pathway in 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 PCNA in all the three human fibroblast cell lines studied, namely normal, xeroderma pigmentosum group A (XP-A) and Cockayne syndrome group B (CS-B). In all the cell lines, induction of PCNA and repair patches occurred in a dose- and time-dependent fashion. Induction of repair patches in NER-deficient XP-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 cells with DNA polymerase inhibitors aphidicolin and cytosine arabinoside. Interestingly, inhibition of repair resynthesis did not affect the intensity of PCNA staining in X-irradiated cells indicating that the PCNA may be required for the BER pathway at a step preceding the resynthesis step.
2 To whom correspondence should be addressed. Tel: +31 715276164; Fax: +31 715221615; Email: natarajan{at}lumc.nl
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