Binding of tsHMG, a mouse testis-specific HMG-domain protein, to cisplatin-DNA adducts.
The anticancer drug cisplatin is particularly effective against testicular tumors. Although the clinical consequences of cisplatin chemotherapy are well-known, the precise mechanism of action remains elusive. Specific recognition of cisplatin-damaged DNA by a class of proteins containing the high-mobility group (HMG) domain DNA-binding motif could play a role in mediating the cytotoxicity of the drug. This study presents a quantitative investigation of binding of the murine testis-specific high-mobility group protein tsHMG to DNA modified by cisplatin. The binding affinity and specificity of this protein to a site-specific 1,2-d(GpG) cisplatin-DNA intrastrand cross-link in a 20 bp probe were determined. A value for the apparent dissociation constant, Kd(app), of 24 +/- 5 nM was obtained by gel mobility shift assays. Binding competition assays with the corresponding unmodified 20 bp probe gave a ratio (rho) of nonspecific to specific Kd(app) values of 230. A polypeptide containing tsHMG domain A (residues 1-82) was expressed and purified to homogeneity. This domain alone was sufficient for specific recognition of cisplatin-modified DNA with a Kd(app) of 300 +/- 50 nM and a rho of 20, a comparatively high discrimination factor. DNase I interference analysis of the adduct-containing strand revealed that tsHMG binding extends over 14 nucleotides, centered around the platinated bases. The domain A polypeptide protection pattern covers a slightly smaller area of 13 nucleotides. The binding affinity and specificity of tsHMG for cisplatin-modified DNA are exceptional compared to those of other HMG-domain proteins studied previously. The possible relevance of these findings to the mechanism of action of cisplatin is discussed.
Ohndorf
U M
UM
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Whitehead
J P
JP
Raju
N L
NL
Lippard
S J
SJ
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D002945:Cisplatin / Q000378:metabolism* / Q000494:pharmacology; D003432:Cross-Linking Reagents; D018736:DNA Adducts / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D000970:Antineoplastic Agents; D003432:Cross-Linking Reagents; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D015226:Dinucleoside Phosphates; D006609:High Mobility Group Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C081930:Tfam protein, mouse; C057270:cisplatin-DNA adduct; C094059:cisplatin-deoxy(guanosine monophosphate guanosine) adduct; D002945:Cisplatin