A clickable psoralen to directly quantify DNA interstrand crosslinking and repair

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• 作者：Benjamin J. Evison ; Marcelo L. Actis ; Naoaki Fujii ; ^{naoaki.fujii@stjude.org" class="auth_mail" title="E-mail the corresponding author}
• 关键词：CSK buffer ; cytoskeleton buffer ; DAPI ; 4&prime ; 6-diamidino-2-phenylindole ; DMSO ; dimethyl sulfoxide ; EDTA ; ethylenediaminetetraacetic acid ; FBS ; fetal bovine albumin ; HEPES ; 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid ; HR ; homologous recombination ; ICL ; interstrand crosslink ; 8-MOP ; 8-methoxypsoralen ; NMR ; nuclear magnetic resonance ; PARP ; poly(ADP-ribose)polymerase ; PBS ; phosphate buffered saline ; PCR ; polymerase chain reaction ; 8-POP ; 8-propargyloxypsoralen ; TAE ; tris-acetate EDTA
• 刊名：Bioorganic & Medicinal Chemistry
• 出版年：2016
• 出版时间：1 March 2016
• 年：2016
• 卷：24
• 期：5
• 页码：1071-1078
• 全文大小：989 K

文摘

DNA interstrand crosslinks (ICLs) represent physical obstacles to advancing replication forks and transcription complexes. A range of ICL-inducing agents have successfully been incorporated into cancer therapeutics. While studies have adopted UVA-activated psoralens as model ICL-inducing agents for investigating ICL repair, direct detection of the lesion has often been tempered by tagging the psoralen scaffold with a relatively large reporter group that may perturb the biological activity of the parent psoralen. Here a minimally-modified psoralen probe was prepared featuring a small alkyne handle suitable for click chemistry. The psoralen probe, designated 8-propargyloxypsoralen (8-POP), can be activated by UVA in vitro to generate ICLs that are susceptible to post-labeling with an azide-tagged fluorescent reporter via a copper-catalyzed reaction. A modified alkaline comet assay demonstrated that UVA-activated 8-POP proficiently generated ICLs in cells. Cellular 8-POP–DNA lesions were amenable to click-mediated ligation to fluorescent reporters in situ, which permitted their detection and quantitation by fluorescence microscopy and flow cytometry. Small molecule DNA repair inhibitors to 8-POP-treated cells attenuated the removal of 8-POP–DNA lesions, validating 8-POP as an appropriate probe for investigating cellular ICL repair. The post-labeling strategy applied in this study is inexpensive, rapid and highly modular in nature with the potential for multiple applications in DNA repair studies.