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 extracts 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 activity was AP sites-dependent and EDTA-sensitive. The unit of enzymatic activity was defined as that amount able to incise 1 ng of plasmid DNA carrying 1 AP site/plasmid at 30 degrees C in 10 min. The assay was used to measure the APE activity in 10 healthy individuals ages 25-48 years. Values ranged from 0.38 to 0.94 units/ng protein, with a mean value 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.
AP endonuclease activity in humans: Development of a simple assay and analysis of ten normal individuals
Magrassi R;
1998
Abstract
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 extracts 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 activity was AP sites-dependent and EDTA-sensitive. The unit of enzymatic activity was defined as that amount able to incise 1 ng of plasmid DNA carrying 1 AP site/plasmid at 30 degrees C in 10 min. The assay was used to measure the APE activity in 10 healthy individuals ages 25-48 years. Values ranged from 0.38 to 0.94 units/ng protein, with a mean value 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
