Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation.
DNA interstrand cross-links are formed by chemotherapy drugs as well as by products of normal oxidative metabolism. Despite their importance, the pathways of cross-link metabolism are poorly understood. Laser confocal microscopy has become a powerful tool for studying the repair of DNA lesions that can be detected by immunofluorescent reagents. In order to apply this approach to cross-link repair, we have synthesized conjugates of 4,5',8-trimethylpsoralen (TMP) and easily detected compounds such as Lissamine rhodamine B sulfonyl chloride (LRB-SC), biotin, and digoxigenin. These conjugates are activated by UVA, and we have analyzed the intracellular localization of DNA damage and DNA reactivity by confocal and immunofluorescence microscopy. The LRB-SC-TMP conjugate 2 appeared mainly in the mitochondria, while the biotin-TMP conjugate 4 preferentially localized in the cytoplasm. Adducts formed by UVA and digoxigenin conjugates of TMP 7a and 4,5'-dimethylangelicin (DMA) 7b, which forms only monoadducts, were largely localized to the nucleus. Exposure of cells incubated with 7a and 7b to a 364 nm UV laser directed toward defined nuclear regions of interest resulted in localized adduct formation which could be visualized by immunofluorescence. Repair-proficient cells were able to remove the photoadducts, while repair-deficient cells were unable to repair the damage. The results indicated that the digoxigenin-TMP conjugate 7a and digoxigenin-DMA conjugate 7b can be used for studying the repair of laser localized DNA monoadducts and cross-links.
Thazhathveetil
Arun Kalliat
AK
Laboratory of Molecular Gerontology, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA.
Liu
Su-Ting
ST
Indig
Fred E
FE
Seidman
Michael M
MM
D000818:Animals; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics; D004076:Digoxigenin / Q000737:chemistry / Q000378:metabolism; D004720:Endonucleases / Q000172:deficiency / Q000235:genetics; D016678:Genome; D006367:HeLa Cells; D006801:Humans; D007834:Lasers; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D014307:Trioxsalen / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D012235:Rhodamines; C027305:Lissamine Rhodamine B sulfonyl chloride; D001710:Biotin; D004247:DNA; C071447:ERCC1 protein, human; D004720:Endonucleases; D004076:Digoxigenin; D014307:Trioxsalen