Histone H3 and heat shock protein GRP78 are selectively cross-linked to DNA by photoactivated gilvocarcin V in human fibroblasts.
Gilvocarcin V (GV) is an antitumor antibiotic with a coumarin-based aromatic structure that promotes protein-DNA cross-linking when photoactivated by near-UV light. We have now identified several proteins that are selectively cross-linked to DNA in human fibroblasts by photoactivated GV, using NH2-terminal amino acid sequencing and Western blot analysis of the purified cross-linked proteins. The selectively cross-linked proteins are histone H3 and GRP78, a heat shock protein belonging to the heat shock protein-70 family. The hydrophobic leader sequence is missing from the cross-linked GRP78, suggesting that only the processed form of the protein is cross-linked to DNA. It is primarily the phosphorylated form of histone H3 that is cross-linked to DNA. Gel retardation analysis from four different GV-treated human fibroblast cell lines revealed two distinct shifted bands, and subsequent immunoblotting confirmed in situ that the slower and the faster bands, respectively, contained GRP78 and histone H3 cross-linked to DNA. The selective cross-linking of these particular proteins is dependent on UV irradiation in the presence of GV, which may help to clarify the unique molecular mechanism of this potent antitumor agent.
Department of Radiation Biophysics and Genetics, School of Medicine, Kobe University, Japan.
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Matsumoto
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Hanawalt
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D003374:Coumarins; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D000091342:Endoplasmic Reticulum Chaperone BiP; D005347:Fibroblasts / Q000187:drug effects; D006027:Glycosides; D006360:Heat-Shock Proteins; D006657:Histones / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D020539:Sequence Analysis, Protein; D014466:Ultraviolet Rays
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D002352:Carrier Proteins; D003374:Coumarins; D003432:Cross-Linking Reagents; D000091342:Endoplasmic Reticulum Chaperone BiP; D006027:Glycosides; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D006657:Histones; D018832:Molecular Chaperones; C031781:gilvocarcin V; D004247:DNA