A simple assay for AP endonuclease activity in human lymphocytes

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• 作者：Redaelli ; Anna ; Magrassi ; Raffaella ; Bonassi ; Stefano ; Abbondandolo ; Angelo ; Frosina ; Guido
• 关键词：AP endonuclease ; Base excision repair ; Peripheral blood lymphocytes
• 刊名：Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
• 出版年：1997
• 出版时间：September, 1997
• 年：1997
• 卷：379
• 期：1
• 页码：S30
• 全文大小：121 K

文摘

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 able to incise one ng of plasmid DNA carrying one AP site/plasmid at 30° C in 10 min. The assay was used to measure the APE activity in ten healthy individuals of ages 25-48 years. Values ranged from 0.38 to 0.94 units/ng protein with a mean value of 0.62. This is the first estimate of the interindividual variability of this important DNA repair enzyme.