BINDING OF TSHMG, A MOUSE TESTIS-SPECIFIC HMG-DOMAIN PROTEIN, TO CISPLATIN-DNA ADDUCTS

The anticancer drug cisplatin is particularly effective against testic ular tumors, Although the clinical consequences of cisplatin chemother apy are well-known, the precise mechanism of action remains elusive. S pecific recognition of cisplatin-damaged DNA by a class of proteins co ntaining the high-mobility group (HMG) domain DNA-binding motif could play a role in mediating the cytotoxicity of the drug, This study pres ents a quantitative investigation of binding of the murine testis-spec ific high-mobility group protein tsHMG to DNA modified by cisplatin. T he binding affinity and specificity of this protein to a site-specific 1,2-d(GpC) cisplatin-DNA intrastrand cross-link in a 20 bp probe were determined. A value for the apparent dissociation constant, K-d(app), Of 24 +/- 5 nM was obtained by gel mobility shift assays. Binding com petition assays with the corresponding unmodified 20 bp probe gave a r atio (rho) of nonspecific to specific K-d(app) values of 230. A polype ptide containing tsHMG domain A (residues 1-82) was expressed and puri fied to homogeneity. This domain alone was sufficient for specific rec ognition of cisplatin-modified DNA with a K-d(app) Of 300 +/- 50 nM an d a rho of 20, a comparatively high discrimination factor. DNase I int erference analysis of the adduct-containing strand revealed that tsHMG binding extends over 14 nucleotides, centered around the platinated b ases. The domain A polypeptide protection pattern covers a slightly sm aller 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 o f these findings to the mechanism of action of cisplatin is discussed.