Purification of PCNA as a nucleotide excision repair protein

Abstract

Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.