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Binding of tsHMG, a mouse testis-specific HMG-domain protein, to cisplatin-DNA adducts
U M Ohndorf1, J P Whitehead, N L Raju
1Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Abstract:
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.
Insights
The testis-specific high-mobility group (tsHMG) protein binds exceptionally well to cisplatin-damaged DNA, suggesting a key role for tsHMG in mediating the anticancer drug's effectiveness against testicular tumors.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Cisplatin is an effective anticancer drug, particularly for testicular tumors, but its precise mechanism of action is not fully understood.
- High-mobility group (HMG) domain proteins are known to bind DNA, and their specific recognition of cisplatin-damaged DNA may contribute to the drug's cytotoxicity.
- The murine testis-specific HMG protein (tsHMG) is investigated for its interaction with cisplatin-modified DNA.
Purpose of the Study:
- To quantitatively investigate the binding affinity and specificity of tsHMG to DNA modified by cisplatin.
- To determine the role of the tsHMG domain A in recognizing cisplatin-DNA adducts.
- To elucidate the binding site and extent of tsHMG interaction with cisplatin-damaged DNA.
Main Methods:
- Gel mobility shift assays were used to determine the apparent dissociation constant (Kd(app)) for tsHMG binding to a site-specific cisplatin-DNA intrastrand cross-link.
- Binding competition assays with unmodified DNA were performed to calculate the ratio of nonspecific to specific binding.
- DNase I interference analysis was employed to map the tsHMG binding site on the cisplatin-modified DNA strand.
Main Results:
- tsHMG exhibited a high binding affinity (Kd(app) = 24 +/- 5 nM) and specificity (ratio (rho) = 230) for cisplatin-modified DNA.
- The tsHMG domain A alone was sufficient for specific recognition of cisplatin-damaged DNA, with a Kd(app) of 300 +/- 50 nM and a rho of 20.
- tsHMG binding covered a 14-nucleotide region centered on the platinated bases, while domain A covered 13 nucleotides.
Conclusions:
- tsHMG demonstrates exceptional binding affinity and specificity for cisplatin-modified DNA compared to other HMG-domain proteins.
- The findings suggest that tsHMG plays a significant role in the mechanism of action of cisplatin, potentially mediating its cytotoxicity in testicular tumors.
- The tsHMG domain A is crucial for the specific recognition of cisplatin-DNA adducts.