Localization of the binding region of high mobility group protein 2 to cisplatin-damaged DNA

Cisplatin (CDDP) is an effective cancer chemotherapeutic drug used in the treatment of several human malignancies. The effectiveness of cisplatin therapy is limited by intrinsic resistance of tumors to this drug as well as the development of secondary tumors, which are also drug resistant. A potential mechanism influencing the sensitivity of cells to CDDP may result from the interaction of specific proteins with CDDP-damaged DNA (CDDP-DNA). In an earlier report, we demonstrated that high mobility group (HMG) proteins 1 and 2 bind with high affinity to CDDP-DNA. In the present study partial proteolytic digestion was used to localize the binding region of HMG2. A proteolytic fragment of approximately 20 kDa, containing the amino-terminal region of the protein, maintains the ability to bind with high affinity to CDDP-DNA, while an amino-terminal fragment of 14 kDa binds with slightly reduced affinity. In contrast, a peptide fragment lacking 51 NH2-terminal amino acids from HMG2 has greatly reduced affinity for damaged DNA. Recombinant peptide fragments containing HMG box 1 or HMG box 2 bind weakly to damaged DNA, while a recombinant fragment containing HMG boxes 1 and 2 binds with high affinity. Hence, our results indicate that the amino-terminal region of HMG2 contains the damaged DNA binding recognition site and that both HMG boxes 1 and 2, present in the parental molecule, are required for high affinity binding of this protein to CDDP-DNA.