Bacteriophage Mu Transposition and the Role of Gyrase Binding Sites

University Of Colorado At Denver, Aurora CO

Investigators

Abstract

Bacterial virus Mu, by virtue of its being both a virus and a transposon, is unusual among transposons in its large size and its high frequency of transposition. Amplification of the 37.2 kb Mu DNA during the virus growth cycle occurs by a series of replicative transposition events which take place within the bacterial nucleoid (DNA and associated proteins), despite the constraints imposed by the complex structure of that body. Interest in the long-range DNA interactions involved in synapsing the ends of prophage DNA within the nucleoid, an early step in the transposition process, led to studies which uncovered a strong gyrase binding site (SGS) in the center of the Mu genome that is required for efficient replication. An earlier model postulated that the SGS promotes synapsis of the Mu prophage termini, and that it does so by organizing the topology of the supercoiled prophage DNA to form a plectonemically interwound loop with the SGS at the apex of the loop and the termini at the base. Experimental tests of predictions of the model have offered strong support for the proposed role of the SGS. While a similar site was found in a second transposing virus, an extensive search found no chromosomal or plasmid sites which could replace the SGS in Mu replication. To determine the unique features of the site, a genetic analysis of the SGS was performed. A region downstream of the "core" of the gyrase site was identified that is responsible for imparting to the SGS the ability to promote Mu replication. This project focuses on two important questions raised by the model: 1) Why is a novel mechanism required for promoting the synapsis of Mu prophage ends; and 2) How does the SGS function in promoting synapsis of the Mu prophage ends? The first question will be addressed by exploring the factors that are responsible for the long delay in Mu replication in the absence of the SGS. A recently developed sensitive assay for detecting the early stages of the first round of transposition after induction of a lysogen will be used to examine factors including the constraints imposed by the domain structure of the bacterial nucleoid, the topological consequences of transcription, and the role of Mu and host encoded proteins. The second question will be addressed by a combined structural and functional analysis of the SGS, with emphasis on the downstream region which imparts to the SGS the ability to promote replication. The analysis will include studies of the binding of gyrase to the SGS and to other gyrase sites and studies on the effects of genetic alterations of the SGS right arm on synapsis of prophage ends. These studies will further understanding of the mechanism of Mu replicative transposition and provide insights into the structure of the bacterial nucleoid (the DNA has to be compacted several thousand fold to fit into the bacterial cell, yet still has to be able to replicate and be transcribed) and the biochemistry of DNA gyrase. Work with this genetically tractable system should provide insight into transposons and viruses of plants and animals that move in and out of their hosts' chromosomes.