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 aromati c 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-t erminal amino acid sequencing and Western blot analysis of the purified cro ss-linked proteins, The selectively cross-linked proteins are histone H3 an d GRP78, a heat shock protein belonging to the heat shock protein-70 family , The hydrophobic leader sequence is missing from the cross-linked GRP78, s uggesting that only the processed form of the protein is cross-linked to DN A, It is primarily the phosphorylated form of histone H3 that is cross-link ed to DNA, Gel retardation analysis from four different GV-treated human fi broblast cell lines revealed two distinct shifted bands, and subsequent imm unoblotting confirmed ill situ that the slower and the faster hands, respec tively; contained GRP78 and histone H3 cross-linked to DNA, The selective c ross-linking of these particular proteins is dependent on UV irradiation in the presence of GV, which may help to clarify the unique molecular mechani sm of this potent antitumor agent.