Mechanisms and models for R-loop formation at a bacteriophage T4 replication origin

The downstream region of the T4 replication origin ori( uvsY) has the propensity to unwind. Hypersensitivity to permanganate has been demonstrated downstream of the origin promoter sequence on the non-template strand within a narrow window of time after a phage infection. The pattern of permanganate sensitivity suggested that a persistent RNA-DNA hybrid or R-loop was located within the origin (Carles-Kinch and Kreuzer, 1997).; The focus of my project was to further analyze the formation and regulation of the RNA-DNA hybrid at the replication origin. The first goal of the project was to develop a topological assay that monitored changes in plasmid supercoiling to directly detect RNA-DNA hybrid formation in vivo. I used E. coli DNA gyrase and T4 topoisomerase II in combination with RNAse H and RNAse A. Next, I wanted to determine the essential DNA sequences required for R-loop formation. I also compared the generation of replicated concatemers of origin sequence mutants with GC inserts and established a relationship between R-loop formation and the replicated products generated by a rolling circle. I established a role for single-stranded binding protein gp32 in R-loop stabilization. My results are consistent with the idea that R-loop formation and stability are finely balanced and depend on the energetics of unwinding.