Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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Ohndorf, U.M. et al.

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.[1]

References

Binding of tsHMG, a mouse testis-specific HMG-domain protein, to cisplatin-DNA adducts. Ohndorf, U.M., Whitehead, J.P., Raju, N.L., Lippard, S.J. Biochemistry (1997) [Pubmed]

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