Detection of the Excised, Damage‐containing Oligonucleotide Products of Nucleotide Excision Repair in Human Cells

Abstract

The human nucleotide excision repair system targets a wide variety of DNA adducts for removal from DNA, including photoproducts induced by UV wavelengths of sunlight. A key feature of nucleotide excision repair is its dual incision mechanism, which results in generation of a small, damage-containing oligonucleotide approximately 24- to 32-nt in length. Detection of these excised oligonucleotides using cell-free extracts and purified proteins with defined DNA substrates has provided a robust biochemical assay for excision repair activity in vitro. However, the relevance of a number of in vitro findings to excision repair in living cells in vivo has remained unresolved. Over the past few years, novel methods for detecting and isolating the excised oligonucleotide products of repair in vivo have therefore been developed. Here we provide a basic outline of a sensitive and versatile in vivo excision assay and discuss how the assay both confirms previous in vitro findings and offers a number of advantages over existing cell-based DNA repair assays. Thus, the in vivo excision assay offers a powerful tool for readily monitoring the repair of DNA lesions induced by a large number of environmental carcinogens and anti-cancer compounds.

Detection of the small oligonucleotide products of nucleotide excision repair in UVB-irradiated human skin

“…Following a 15 min centrifugation at maximum speed in a microcentrifuge at 4°C, the DNA pellets were washed with 1 ml of cold 70% ethanol, centrifuged again for 5 min, and then air dried. The subsequent methods of sedDNA labeling and detection from skin epidermis were similar to that reported for cultured cells [9,10,14,15]. Briefly, the soluble DNAs were resuspended in 50 μl of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA), immunoprecipitated with 5 μl of Dynabead slurry bound to anti-(6-4)PP antibody, washed with Wash Buffer I (20 Mm Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS), Wash Buffer II (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), Wash Buffer III (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 150 mM LiCl, 1% Nonidet P-40, 1% sodium deoxycholate), Wash Buffer IV (100 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM LiCl, 1% Nonidet-P40, 1% sodium deoxycholate), and twice with TE buffer.…”

Impact of Age and Insulin-Like Growth Factor-1 on DNA Damage Responses in UV-Irradiated Human Skin

“…These processes may dilute CPD content within genomic DNA in a DNA repair-independent manner, and this complication therefore affects the accuracy of CPD quantitation as a reliable measure of nucleotide excision repair capacity in the skin. Thus, the application of novel technologies, including assays that directly detect the sedDNA products of nucleotide excision repair [ 101 , 102 , 103 , 104 , 105 ], may be advantageous in the future for quantifying DNA repair capacity as a function of age following UV exposures and for correlating these factors to skin carcinogenic risk.…”

Simultaneous detection of nucleotide excision repair events and apoptosis-induced DNA fragmentation in genotoxin-treated cells

“…The small, soluble repair products are then purified, labeled on the 3′ end with a biotinylated nucleotide, and electrophoresed on a sequencing gel. Following electrophoretic transfer to a nylon membrane, the DNAs can be visualized with streptavidin-bound HRP and chemiluminescent detection 10 . An example of this repair assay in HeLa cells exposed to 20 J/m 2 of UVC and then harvested at various time points following irradiation is shown in Fig.…”

“…Though this phenomenon was initially demonstrated in vitro using defined DNA damage-containing templates and cell-free extracts 3 , 4 or purified repair proteins 5 , 6 , nearly identical observations have subsequently been made using cultured cells exposed to UV or other related environmental carcinogens and anti-cancer compounds. The detection of these repair events in vivo was made possible by the fact that these excised oligonucleotides are readily solubilized during cell lysis, and, once purified, can be labeled, separated by electrophoresis, and detected via radioactivity or chemiluminescence 7 – 10 . Moreover, the fact that these excised DNAs are released from DNA in association with specific repair proteins 7 , 11 has allowed for studies of post-excision repair events 12 , 13 and for the mapping of DNA repair events throughout the genome 2 , 14 – 18 .…”