This proposal focuses on the possible role on transcriptionally-induced structural changes in DNA may have on genetic recombination in E. coli cells. There is much data showing that transcription elevates different types of recombination. Though the mechanisms responsible for this phenomenon are unknown, a popular belief is that changes in DNA elicited during transcription might be involved. The proof of this hypothesis depends on the ability to distinguish between the effects of DNA sequence and DNA structure in the cell. Recently, we and others found that activation of a plasmid promoter in E. coli cells increases the upstream supercoiling, driving the formation of such non-B DNA structures as cruciforms, Z-DNA and triplexes. We plan to use this system for analyzing the influence of transcriptionally-induced unusual DNA structures on recombination. Specifically, we will study transcriptionally-driven DNA supercoiling and the formation of non-B DNA structures in vivo using chemical probing of intracellular DNA with reagents specific to single-stranded DNA bases. We will then analyze the influence of these structures on homologous recombination using an E.coli system with two plasmids carrying distinct mutations in a tet gene under the control of an inducible promoter. Quite recently we also found that some simple repeats, which potentially form unusual DNA structures, tend to delete from bacterial plasmids when incorporated downstream from the transcriptional start site. The promoter's activity and the orientation of the repeat in the transcribing unit caused an all-or-none affect on repeats maintenance. To understand the mechanisms of repeats instability caused by transcription we will analyze whether deletion formation is due to the passive selection of plasmids with shorter inserts or to active rearrangements involving RNA polymerase. We will characterize the effects of transcription through repeated sequences on the growth and nucleic acids metabolism of E. coli cells. Finally, we will examine the interaction of RNA polymerase with repeated sequences in vitro and in vivo. To this end we will locate RNA polymerase pause sites and dead-end complexes within "unstable" repeats in vitro and attempt to isolate complexes between RNA polymerase and repeat-containing plasmids from E. coli cells. The long-term goal of this proposal is to prove that transcriptionally- induced changes in DNA secondary structure could affect different types of genetic recombination.
NIH ExPorter
NIH ExPorter