The experiments proposed in this renewal application revolve around three primary goals. The first is to design an enzyme for making positively supercoiled DNA. It is based on the thesis that during transcription a positive superhelical debt is built ahead of the replication fork while a negative superhelical tension accumulates behind the replication fork. The situation is dramatic when both the DNA and polymerase are constrained during the act of transcription. The model for such an enzyme is a fusion protein that generates both conditions, that is, a protein containing a sequence-specific DNA binding domain joined to RNA polymerase. The second goal is to analyze chromatin structure in vivo. DNA nucleases will be cloned under the control of inducible regulatory sequences in yeast and in higher eukaryotic cells. To obviate problems in making nucleases in vivo, the strategy is to construct cold sensitive mutants that are inactive at low temperature, but able to cleave DNA in vivo when the medium is shifted to a higher temperature. The third goal is to make supercoiled RNA. The results of the research should enhance our understanding of the structure of genes and how they function at the molecular level in living systems.
