title: A clickable psoralen to directly quantify DNA interstrand crosslinking and repair.
abstract: 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.
authors.affiliation: Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Evison
authors.affiliation: Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
authors.forename: Marcelo L
authors.initials: ML
authors.lastname: Actis
authors.affiliation: Department of Chemical Biology and Therapeutics, St Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA. Electronic address: naoaki.fujii@stjude.org.
authors.forename: Naoaki
authors.initials: N
authors.lastname: Fujii
mesh_terms: D000480:Alkynes / Q000737:chemistry; D001386:Azides / Q000737:chemistry; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D018736:DNA Adducts / Q000032:analysis*; D004260:DNA Repair; D005363:Ficusin / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D014466:Ultraviolet Rays
chemical_list: D000480:Alkynes; D001386:Azides; D018736:DNA Adducts; D005456:Fluorescent Dyes; D003300:Copper; D005363:Ficusin
keywords: Chemical screening; Click chemistry; DNA repair; Imaging; Interstrand DNA crosslink; Psoralen
references.citation: Mutat Res. 2001 Sep 4;486(4):217-47
references.pmid: 11516927
references.citation: J Org Chem. 2010 Jul 2;75(13):4644-7
references.pmid: 20527977
references.citation: Mol Biotechnol. 2004 Mar;26(3):249-61
references.pmid: 15004294
references.citation: Chem Biol. 2004 Apr;11(4):535-46
references.pmid: 15123248
references.citation: Proc Natl Acad Sci U S A. 1978 Jun;75(6):2703-7
references.pmid: 275838
references.citation: J Biochem. 1983 Oct;94(4):1259-67
references.pmid: 6654857
references.citation: J Biochem. 1984 May;95(5):1253-7
references.pmid: 6746605
references.citation: Natl Cancer Inst Monogr. 1984 Dec;66:21-30
references.pmid: 6531031
references.citation: Nucleic Acids Res. 1985 Apr 11;13(7):2533-44
references.pmid: 4000963
references.citation: Annu Rev Biochem. 1985;54:1151-93
references.pmid: 2411210
references.citation: Cancer Res. 1987 May 1;47(9):2451-5
references.pmid: 2436765
references.citation: Cancer Res. 1988 Sep 1;48(17):4762-5
references.pmid: 2842037
references.citation: Nucleic Acids Res. 1989 Sep 12;17(17):6915-26
references.pmid: 2780314
references.citation: J Invest Dermatol. 1991 Jul;97(1):151-5
references.pmid: 2056186
references.citation: Carcinogenesis. 1993 Jul;14(7):1303-11
references.pmid: 8392444
references.citation: J Chromatogr. 1993 Aug 25;618(1-2):277-88
references.pmid: 8227260
references.citation: Biochemistry. 1995 Oct 10;34(40):12937-53
references.pmid: 7548052
references.citation: J Org Chem. 2011 Jan 21;76(2):450-9
references.pmid: 21175145
references.citation: Genes Dev. 2011 Mar 1;25(5):409-33
references.pmid: 21363960
references.citation: Nat Rev Cancer. 2011 Jul;11(7):467-80
references.pmid: 21701511
references.citation: J Biol Chem. 2012 Apr 20;287(17):14289-300
references.pmid: 22383522
references.citation: Chem Res Toxicol. 2012 Aug 20;25(8):1609-15
references.pmid: 22621314
references.citation: Angew Chem Int Ed Engl. 2013 Mar 18;52(12):3350-4
references.pmid: 23427109
references.citation: Anal Chem. 2013 Jul 16;85(14):6732-9
references.pmid: 23789926
references.citation: J Am Chem Soc. 2013 Aug 14;135(32):11680-3
references.pmid: 23879391
references.citation: J Vis Exp. 2010;(41). pii: 1958. doi: 10.3791/1958
references.pmid: 20689508
references.citation: Cold Spring Harb Perspect Biol. 2013 Oct;5(10):a012732
references.pmid: 24086043
references.citation: Mol Cell. 2013 Nov 7;52(3):434-46
references.pmid: 24207054
references.citation: Ann Oncol. 2014 Jan;25(1):32-40
references.pmid: 24225019
references.citation: J Biol Inorg Chem. 2014 Mar;19(3):415-26
references.pmid: 24407462
references.citation: J Biol Chem. 2014 Mar 7;289(10):7109-20
references.pmid: 24474685
references.citation: ACS Chem Biol. 2014 Oct 17;9(10):2404-11
references.pmid: 25055168
references.citation: Nucleic Acids Res. 2014 Dec 16;42(22):14013-21
references.pmid: 25429975
references.citation: Science. 2015 May 1;348(6234):1253671
references.pmid: 25931565
references.citation: Bioorg Med Chem. 2015 Nov 1;23(21):6912-21
references.pmid: 26462050
references.citation: Bioconjug Chem. 2007 Mar-Apr;18(2):431-7
references.pmid: 17373769
references.citation: Anal Chem. 2008 Nov 15;80(22):8790-8
references.pmid: 18947205
references.citation: Cancer Sci. 2008 Nov;99(11):2238-43
references.pmid: 18771529
references.citation: Cell. 2009 Feb 6;136(3):473-84
references.pmid: 19203582
references.citation: Mutagenesis. 2009 Mar;24(2):173-81
references.pmid: 19147795
references.citation: J Biol Chem. 2009 Oct 9;284(41):27908-17
references.pmid: 19684342
references.citation: J Am Chem Soc. 2003 Apr 23;125(16):4686-7
references.pmid: 12696868