DNA transposition, the "hopping" of DNA sequences into new chromosomal loci is now widely recognized to be important in a wide variety of biological phenomena, including the evolution of drug resistance plasmids and the mechanism of carcinogenesis. The biochemical pathway of transposition remains largely undefined. An in vitro replicative transposition system has been established for the bacteriophage Mu, a transposable element which achieves the highest frequency of transposition known in any biological system. Since it is now possible to analyze a Mu transposition event in vitro in which every element is hopping from a single chromosomal "donor site", the following questions with respect to the biochemical pathway for transposition will be addressed in the next grant period. Since Mu replicative transposition is semi-conservative, which of the parental Mu-host connections are broken, and what new connections are made? What is the order of breaking and making connections with respect to replicating Mu sequences? Are there covalent protein-DNA complexes involved in breaking and making these connections? A second set of studies has as a long-term goal to understand the role of each Mu component in the transposition process. An initial aim is to develop functional assays for, and to purify the MuA gene product. In addition, since only a limited number of Mu sequences are transposing during the Mu lytic cycle, experiments will be carried out to determine how the choice is made as to which of the available sequences will hop. Finally, the proportion of intrachromosomal transposition events which are simple insertions, and which are of the cointegrate type will be estimated. Experiments on three purified enzymes, DNA polymerase I, poly ADP-ribose synthetase and DNA topoisomerase I will be carried out. The goals with respect to DNA polymerase I involve elucidating the mechanism of generating "foldback" DNA during nick translation, and the effect of displaced single stranded structures on general recombination. Finally, a modification reaction recently discovered, the poly ADP-ribosylation of eukaryotic DNA topoisomerase I will be characterized.
NIH ExPorter
NIH ExPorter