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.
Matsumoto
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Department of Radiation Biophysics and Genetics, School of Medicine, Kobe University, Japan.
Hanawalt
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PC
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