Most naturally occurring DNA exists as a B-helix (a right-handed helix). Among other documented modifications, DNA may become methylated and may possess regions of the left-handed Z-helix. Methylated DNA and Z-DNA have been separately implicated in gen regulation. The proposed research will provide system for the in vitro examination of new methylation and Z-DNA formation affect on specific DNA function, transcription. Further, since DNA methylation is known to promote Z-DNA formation, the interplay between DNA methylation, Z-DNA formation and gene control will be examined. In order to understand the molecular details of these processes, oligonucleotides containing potential Z-DNA sequences flanked by sequences which would remain in the right-handed B conformation will be synthesized. The structures of the junctions between the Z segments and B segments will be examined using 1H NMR, digestion with enzymes that select for unusual DNA structures, and reaction with small molecules which bind preferentially to particular sequence and/or structure types. A tenet of the hypothesis of this research is that methylases may preferentially target DNA segments that can flip to Z structures by recognizing the """"""""proto-junctions"""""""" existing between potential Z segments and flanking sequences. Thus the """"""""proto-junctions"""""""" of oligomers in methylated potential Z segments will be examined with NMR, enzyme digestion and small molecule binding. Poly junction molecules, made by ligating these oligomers will serve as substrates for methylase binding, methylase activity, RNA polymerase binding and RNA polymerase activity studies. Enzyme digestion and small molecule reaction studies will provide data suggesting that the junction and proto-junction structures studied in the oligomers occur in these fragments. Following the sequential course of methylase binding, methylation and polymerase activity in DNA containing proto-junctions will give the interrelationship between these functions. The proposed research will provide training for students in a broad range of biochemical areas. All students will be involved in DNA synthesis, DNA sequencing, cloning, enzymatic assays, spectroscopic analyses of DNA structures and drug binding studies. Exposing students to many and varied techniques shall enable them to move into a wide scope of research areas.
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