Binding Of High Mobility Group Protein Research Articles

Interactions between HMG proteins (HMG1/2 and HMG14/ 17) and human ?-globin gene promoter (?-promoter)

High mobility group (HMG) proteins are abundant non-histone proteins in the nuclei of eukaryocytes. It has been shown that HMG proteins may play important roles in the structure and function of chromatin. In the present study, the binding of HMG proteins (HMG1/2 and HMG14/17) to the human e-globin gene promoter (e-promoter, −177–+1 bp) has been examined by using both the in vitro nucleosome reconstitution and the electrophoresis mobility shift assay (EMSA). We found that HMG1/2 proteins could bind to the naked e-promoter DNA, however, HMG14/17 could not. Using the in vitro nucleosome reconstitution, we revealed that HMG14/17 could bind to the mononucleosome reconstituted in vitro with e-promoter, while HMG1/2 could not. Those results indicate that the binding of HMG proteins to e-promoter is dynamic as the nucleosome assembling and disassembling. We speculated that this selective binding of HMG proteins to e-promoter might play a critical role in the regulation of e-globin gene expression.

Effect of pH on interactions between DNA and high-mobility group protein HMG1.

Fluorescence quenching was used to test the effect of pH changes on the binding of high-mobility group protein 1 (HMG1) to double-stranded and single-stranded DNA. At pH 7.5, the binding constant K for double-stranded DNA was 3 x 10(6) M-1, the binding site size n was 13, and the cooperativity factor q was 78, while at pH 6 the corresponding values were K = 12 x 10(6) M-1, n = 54, and q = 770. For the binding of HMG1 to single-stranded DNA at pH 7.5, the values were K = 2 x 10(6) M-1, n = 7, and q = 60, whereas at pH 6 they were K = 3 x 10(6) M-1, n = 14, and q = 440. Denaturation of HMG1 by oxidation of its sulfhydryl groups substantially affected the binding parameters. At pH 6, double-stranded DNA bound oxidized HMG1 with K = 6 x 10(6) M-1, n = 16, and q = 200, and single-stranded DNA bound with K = 3 x 10(6) M-1, n = 7, and q = 180. The sensitivity of the double-stranded DNA-HMG1 interaction to pH, along with an earlier report of a sharp optimum of binding at 140 mM NaCl, reveals a potential for in vivo regulation of the strength and mode of HMG1 binding by DNA through the action of analogous factors in the cellular milieu.

Characterization of high mobility group protein binding to cisplatin-damaged DNA

cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) is a widely used chemotherapeutic agent. While many tumors are highly responsive to CDDP, certain tumors are resistant to this drug, limiting its efficacy. The anti-tumor activity of CDDP is believed to result from its coordination bonding to chromosomal DNA. Alterations in tumor cell sensitivity to CDDP may result from the presence or absence of protein(s) which specifically recognize CDDP-damaged DNA. We have developed a damaged-DNA affinity precipitation assay that allows the direct identification of cellular proteins that bind to CDDP-damaged DNA. Using this procedure, we have identified several proteins which specifically bind to CDDP-damaged DNA. Two of these proteins have been identified as high mobility group proteins (HMG) 1 and 2 In the current report, we have characterized the binding of these proteins to CDDP-DNA. The calculated Kd of binding to CDDP-damaged DNA was 3.27 × 10 −10 for HMG1 and 1.87 × 10 −10 for HMG2. Using highly specific chemical modifying reagents, we have determined that Cys residues play an important role in protein binding. We also observed that HMG2 will bind to DNA modified with carboplatin and iproplatin although to a lesser extent than to DNA damaged with CDDP. Thus, our results indicate that HMG 2 binds with high affinity to DNA modified with therapeutically active platinum compounds. In addition, our findings suggest that thiol groups play an essential role in the binding of HMG1 and HMG2 to CDDP-DNA.