AP ENDONUCLEASE ACTIVITY IN HUMANS - DEVELOPMENT OF A SIMPLE ASSAY AND ANALYSIS OF 10 NORMAL INDIVIDUALS

The human AP endonuclease (APE) activity counteracts the mutagenic and cytotoxic effects of the frequent genomic lesions abasic (AP) sites. In order to investigate the interindividual variability of APE levels, a simple and quantitative assay was developed. Crude lymphocyte extra cts were incubated with a depurinated or a control supercoiled plasmid substrate, and the accumulation of nicked circular plasmid forms was monitored by agarose gel electrophoresis. The detected incision activi ty was AP sites-dependent and EDTA-sensitive. The unit of enzymatic ac tivity was defined as that amount able to incise 1 ng of plasmid DNA c arrying 1 AP site/plasmid at 30 degrees C in 10 min. The assay was use d to measure the APE activity in 10 healthy individuals ages 25-48 yea rs. Values ranged from 0.38 to 0.94 units/ng protein, with a mean valu e of 0.62. The use of the assay for screening of people with DNA base excision repair (BER) defects is proposed. (C) 1998 Wiley-Liss, Inc.