The presence of a non-B-Z-structure enhances an enzyme's reactivity at its recognition site

Alternating (CG) sequences form an unusual conformation in the presence of cobalt hexamine. The oligomer, BZ-IV, containing a (CG)4 run (BZ-IV sequence: 5'TCGACGCGCGCGATCAGTCA- 3') was inserted at the Sal I site of the Escherichia coli pGEM-5zf(+) plasmid producing the plasmid pCW001. Hinf I digestion of pCW001 produced a 367 base pair (bp) fragment containing the BZ-IV insert. For controls, the 452 bp Hinf I fragment from the pCW001 plasmid and the 347 bp Hinf I fragment from the pGEM plasmid were used. Digestion studies were performed using the restriction enzymes Bgl I, EcoRV, Hha I, Mbo I, Not I, Pst I, and Taq I and methylation studies were performed using dam methylase. Data were obtained by beta scanning or ethidium bromide staining the polyacrylamide gels of the digestion or methylation products. The results show that in the presence of 100 uM cobalt hexamine, in which BZ-IV takes on a non-B-Z-structure, the enzyme's ability to react and cleave its recognition site is enhanced.

Binding of small molecules to DNA junctions

Previous results in our laboratory suggest that the (CG) 4 segments whether present in a right-handed or a left-handed conformation form distinctive junctions with adjacent random sequences. These junctions and their associated sequences have unique structural and thermodynamic properties that may be recognized by DNA-binding molecules. This study probes these sequences by using the following small ligands: actinomycin D, 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione, ametantrone, and tris(phenanthroline)ruthenium (II). These ligands may recognize the distinctive features associated to the (CG)4 segment and its junctions and thus interact preferentially near these sequences. Restriction enzyme inhibition assays were used to determine whether or not binding interactions took place, and to approximate locations of these interactions. These binding studies are first carried out using two small synthetic oligomers BZ-III and BZ-IV. The (5meCG)4 segment present in BZ-III adopts the Z-conformation in the presence of 50 m M Co(NH3)63+. In BZ-IV, the unmethylated (CG)4 segment changes to a non-B conformation in the presence of 50 m M Co(NH3)63+. BZ-IV, containing the (CG)4 segment, was inserted into a clone plasmid then digested with the restriction enzyme Hinf I to produce a larger fragment that contains the (CG)4 segment. The results obtained on the small oligomers and on the larger fragment for restriction enzyme Mbo I indicate that 1,4-bis(((di(aminoethyl)amino)ethyl)amino)anthracene-9,10-dione binds more efficiently at or near the (CG)4 segment. Restriction enzymes EcoRV, Sac I and Not I with cleavage sites upstream and downstream of the (CG)4 insert were used to further localize binding interactions in the vicinity of the (CG)4 insert. RNA polymerase activity was studied in a plasmid which contained the (CG)4 insert downstream from the promoter sites of SP6 and T7 RNA polymerases. Activities of these two polymerases were studied in the presence of each one of the ligands used throughout the study. Only actinomycin D and spider, which bind at or near the (CG)4 segment, alter the activities of SP6 and T7 RNA polymerases. Surprisingly, enhancement of polymerase activity was observed in the presence of very low concentrations of actinomycin D. These results suggest that the conformational features of (CG) segments may serve in regulatory functions of DNA. ^