Repair of oxidative damage of thymine by HeLa whole-cell extracts: simultaneous analysis using a microsupport and comparison with traditional PAGE analysis

Abstract

In mammalian cells, the base excision repair (BER) pathway allows the remove of small DNA base lesions such as oxidized bases. It is initiated by glycosylases that removed the modified base leaving an abasic site that is subsequently processed by AP endonuclease activities. Measurement of BER activities in cell extracts is time consuming and hazardous when radioactive material is used. We report in this study, the parallelized fluorescent analysis of excision of several oxidation products of thymine by cell extracts. To conduct the study, 5-(hydroxymethyl)uracil, 5-formyluracil, 5-carboxyuracil and formylamine together with uracil and the control thymine, were incorporated into oligonucleotides of identical sequences and paired either with adenine or with guanine containing DNA fragments. The oligonucleotides were fixed by sandwich hybridization in wells of a microplate (OLISA technology). Excision by HeLa extracts of the six different DNA base lesions could be followed simultaneously in the same well. Our results showed that the extent of excision of the lesions was the same on support and in solution using classical PAGE analysis approach with modified 32P-labeled oligonucleotides. We demonstrated that the simultaneous analysis on support is a successful approach to facilitate high-throughput screening of BER activities present in cell extracts. Moreover, extended study of 5-carboxyuracil revealed that this lesion displays similar biological properties as 5-formyluracil.