Highly specific and sensitive method for measuring nucleotide excision repair kinetics of ultraviolet photoproducts in human cells

Abstract

The nucleotide excision repair pathway removes ultraviolet (UV) photoproducts from the human genome in the form of short oligonucleotides ∼30 nt in length. Because there are limitations to many of the currently available methods for investigating UV photoproduct repair in vivo, we developed a convenient non-radioisotopic method to directly detect DNA excision repair events in human cells. The approach involves extraction of oligonucleotides from UV-irradiated cells, DNA end-labeling with biotin and streptavidin-mediated chemiluminescent detection of the excised UV photoproduct-containing oligonucleotides that are released from the genome during excision repair. Our novel approach is robust, with essentially no signal in the absence of UV or a functional excision repair system. Furthermore, our non-radioisotopic methodology allows for the sensitive detection of excision products within minutes following UV irradiation and does not require additional enrichment steps such as immunoprecipitation. Finally, this technique allows for quantitative measurements of excision repair in human cells. We suggest that the new techniques presented here will be a useful and powerful approach for studying the mechanism of human nucleotide excision repair in vivo.

An Integrated Approach for Analysis of the DNA Damage Response in Mammalian Cells

“…The reactions were subsequently extracted with phenol/chloroform precipitated in ethanol. After centrifugation, the pellets were washed with 70% ethanol, resuspended in TE buffer, and then subjected to labeling with terminal deoxynucleotidyl transferase and the nucleotide analog biotin-11-dUTP as described previously with a few modifications (7). Briefly, the extracted DNA was incubated with 20 units of terminal deoxynucleotidyl transferase (New England Biolabs), 10 M of biotin-11-dUTP (Biotium, Inc.), and 0.25 mM CoCl 2 in 50 l of 1ϫ Terminal Transferase Reaction Buffer (New England Biolabs) for 30 min at 37°C.…”

“…Recently, we developed a method for quantifying the repair of UV-induced DNA damage in vivo with high sensitivity, which can detect repair within minutes of UV irradiation (7,8). The method consists of observing the nucleotide excision repair-specific ϳ30-nt-long oligomers ("nominal 30-mers," which are actually in the range of 24 -32 nucleotides) that had previously only been visualized in vitro (9 -12).…”

“…The method consists of observing the nucleotide excision repair-specific ϳ30-nt-long oligomers ("nominal 30-mers," which are actually in the range of 24 -32 nucleotides) that had previously only been visualized in vitro (9 -12). The method involves cell lysis followed by immunoprecipitation of the nominal 30-mer with antibodies against the repair factor TFIIH which is bound to the oligomer or with antibodies against cyclobutane pyrimidine dimers (CPDs) 4 or (6 -4) photoproducts [(6 -4)PPs] and labeling of the excised oligonucleotide with either radioisotope or non-radioisotopic methods (7,8,13). The approach in its original form has been quite useful for characterizing DNA repair in mammalian cells in vivo (7,8,13) and also for mapping excision repair events genome-wide (14) but has so far only been used for studying repair of UVinduced DNA damage.…”

“…The method involves cell lysis followed by immunoprecipitation of the nominal 30-mer with antibodies against the repair factor TFIIH which is bound to the oligomer or with antibodies against cyclobutane pyrimidine dimers (CPDs) 4 or (6 -4) photoproducts [(6 -4)PPs] and labeling of the excised oligonucleotide with either radioisotope or non-radioisotopic methods (7,8,13). The approach in its original form has been quite useful for characterizing DNA repair in mammalian cells in vivo (7,8,13) and also for mapping excision repair events genome-wide (14) but has so far only been used for studying repair of UVinduced DNA damage. Because nucleotide excision repair operates on essentially all types of DNA lesions, in particular on the so-called "bulky DNA adducts" (4, 5), we suggested that with appropriate modifications the method could also be used for investigating other DNA damage repaired by nucleotide excision repair as well.…”

Human genome-wide repair map of DNA damage caused by the cigarette smoke carcinogen benzo[a]pyrene

“…BPDE preferentially forms bulky covalent DNA adducts at N2 position of guanines and causes mutations if these BPDE-deoxyguanosines (BPDE-dGs) are not efficiently eliminated by nucleotide excision repair (3). Various methods of varying resolutions have been developed for mapping DNA damage and repair genome-wide (4)(5)(6)(7)(8)(9). We previously reported a method, termed excision repair-sequencing (XR-seq), for mapping nucleotide excision repair (6).…”