Protection of all cleavable sites of DNA with the multiple CGCG or continuous CGG sites from the restriction enzyme, indicative of simultaneous binding of small ligands.

The anthracenone ligands (1-12) with a keto-phenol and a hydroxamic acid unit were synthesized and evaluated by a restriction enzyme inhibition assay. DNA substrates composed of multiple CGCG or CGG sites are fully hydrolyzed by a restriction enzyme that is selective for each sequence. Under such conditions, the full-length DNA substrate remains only when the ligand binds to all binding sites and protects it from hydrolysis by the restriction enzymes. In the assay using AccII and the 50-mer DNA substrates containing a different number of CGCG sites at different non-binding AT base pair intervals, the more the CGCG sites, the more the full-length DNA increased. Namely, simultaneous binding of the ligand (5) to the CGCG sites increased in the order of (CGCG)5>(CGCG)2>(CGCG)1. Furthermore, the length of the spacer of the hydroxamic acid to the anthracenone skeleton played an important role in the preference for the number of the d(A/T) base pairs between the CGCG sites. The long spacer-ligand (5) showed a preference to the CGCG sites with five AT pairs, and the short spacer-ligand (10) to that with two AT pairs. The ligand (12) with the shortest spacer showed a preference in simultaneous binding to the 54-mer DNA composed of 16 continuous CGG sites in the assay using the restriction enzyme Fnu4HI that hydrolyzes the d(GCGGC)/d(CGCCG) site. Application of these ligands to biological systems including the repeat DNA sequence should be of significant interest.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg2+, which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat.