PCR-BASED DEVELOPMENT OF DNA SUBSTRATES CONTAINING MODIFIED BASES - AN EFFICIENT SYSTEM FOR INVESTIGATING THE ROLE OF THE EXOCYCLIC GROUPS IN CHEMICAL AND STRUCTURAL RECOGNITION BY MINOR-GROOVE BINDING-DRUGS AND PROTEINS

DNA molecules containing inosine in place of guanosine and/or 2,6-diam inopurine in place of adenine have been synthesized and tested as subs trates for binding of sequence-selective ligands, both small and large . Footprinting patterns reveal that the binding sites for AT- or CC-sp ecific antibiotics (distamycin or mithramycin, respectively) are compl etely changed in the modified DNAs, as expected for direct sequence re adout involving contact with the purine 2-amino group. However, we als o find large changes in the binding of HMG-D, a member of the HMG-1 fa mily of chromosomal proteins, pointing to an indirect influence of the exocyclic amino group on ligand binding via an effect on the deformab ility of the double helix, This interpretation is confirmed by the fin ding that deoxyuridine-containing poly- and oligonucleotides, which la ck the exocyclic methyl group of thymidine in the major groove, intera ct 5-10 times more strongly with HMG-D than do their counterparts cont aining natural nucleotides.