Analysis of nucleotide excision repair by detection of single-stranded DNAtransients

Nucleotide excision repair (NER) removes bulky DNA lesions and is thus cruc ial for the protection against environmental carcinogens and UV light expos ure. Deficiencies in NER cause increased mutation rates and chromosomal abe rrations. Current methods for studying NER are mostly based on either quant itation of lesion removal or detection of repair DNA synthesis. Both have t heir limitations: lesion removal is inaccurate at very short times post-les ion, where the fraction of removal is low. Repair synthesis is difficult to apply to normally cycling cells due to the need to discriminate repair fro m replicative DNA synthesis. To overcome these problems we developed a meth od for analysis of NER based on detection of transient single-stranded (ss) DNA stretches generated at the nucleotide excision step. Cells are metabol ically labelled with BrdU, exposed to UV-irradiation and the ssDNA transien ts generated during excision repair are detected using an anti-BrdU antibod y. The method allows single-cell microscopic analysis of the distribution o f DNA repair sites as well as kinetic analysis of the DNA repair response. Studies using various DNA repair-deficient cell lines indicate that the det ection method integrates a number of pre-synthesis nucleotide excision repa ir stages. Thus, assembled repair sites can be detected even when they may not lead to complete resolution of the DNA lesion. Using this approach, we show that repair helicase-deficient cells differ from endonuclease-deficien t cells.