Abstract
Novel in vivo excision assays for monitoring the excised oligonucleotide products of nucleotide excision repair in UV-irradiated cells have provided unprecedented views of the kinetics and genomic distribution of repair events. However, an unresolved issue is the fate of the excised oligonucleotide products of repair and their mechanism of degradation. Based on our observation that decreases in excised oligonucleotide abundance coincide with the induction of apoptotic signaling in UV-irradiated cells, we considered the possibility that caspase-mediated apoptotic signaling contributes to excised oligonucleotide degradation or to a general inhibition of the excision repair system. However, genetic and pharmacological approaches to inhibit apoptotic signaling demonstrated that caspase-mediated apoptotic signaling does not affect excision repair or excised oligonucleotide stability. Nonetheless, our assay for detecting soluble DNAs produced by repair also revealed the production of larger DNAs following DNA damage induction that was dependent on caspase activation. We therefore further exploited the versatility of this assay by showing that soluble DNAs produced by both nucleotide excision repair and apoptotic signaling can be monitored simultaneously with a diverse set of DNA damaging agents. Thus, our in vivo excision repair assay provides a sensitive measure of both repair kinetics and apoptotic signaling in genotoxin-treated cells.
Highlights
The human nucleotide excision repair system removes a wide variety of lesions from DNA through a dual incision mechanism, in which the damaged nucleotides are essentially cut out of the DNA in the form of a small DNA oligonucleotide approximately 30 nt in length[1,2]
Based on our observation that the induction of apoptotic signaling was correlated with a decrease in excised oligonucleotide abundance in cultured cells following UV exposure, we tested the hypothesis that nucleotide excision repair and/or excised oligonucleotide stability were directly regulated by caspase-mediated apoptotic signaling
Because we previously showed that excised oligonucleotide products of repair could be observed in cells treated with these compounds[9], we quantified the level of excised oligonucleotides, cleaved PARP, and large, soluble DNAs as a function of time in cells treated with BPDE and cisplatin
Summary
The human nucleotide excision repair system removes a wide variety of lesions from DNA through a dual incision mechanism, in which the damaged nucleotides are essentially cut out of the DNA in the form of a small DNA oligonucleotide approximately 30 nt in length[1,2] 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. This discovery demonstrates the versatility of our assay for monitoring soluble DNAs that are generated in genotoxin-treated cells and should facilitate future studies of DNA damage responses in human cells exposed to a wide variety of DNA damaging agents
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