Nature of full-length HMGB1 binding to cisplatin-modified DNA.
HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K(d) of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.
Jung
Yongwon
Y
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Lippard
Stephen J
SJ
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002945:Cisplatin / Q000737:chemistry / Q000494:pharmacology*; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D024243:HMGB1 Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007364:Intercalating Agents / Q000737:chemistry / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics
D018736:DNA Adducts; D004268:DNA-Binding Proteins; D024243:HMGB1 Protein; D007364:Intercalating Agents; D010446:Peptide Fragments; D010649:Phenylalanine; D000409:Alanine; D002945:Cisplatin