The movement of transposable elements generates a variety of genetic rearrangements that can profoundly affect the genetic program of their host cells. The Harshey lab is studying phage Mu as a model transposon, whose transposition mechanism displays similarities to the integration mechanism of retroviral DNA. The chemical steps of Mu transposition are carried out within a high order DNA-protein assembly in which the transposase (MuA protein) assumes a tetrameric configuration. Organization of this tetrameric complex is highly regulated, requiring several co-factors including an enhancer element. Recent results from the Harshey and other laboratories suggest that the monomeric form of MuA carries only a partial active site. Full active sites are assembled by sharing residues between separate MuA monomers within the tetramer. A major goal of this proposal is to test models for active site assembly. Strategies are proposed to determine which combinations of subunits within the MuA tetramer assemble into active sites that function in transposon end cleavage and in subsequent DNA strand exchange. In addition, experiments are designed to understand the role of the enhancer element in directing the MuA assembly process. Several site-specific DNA recombination systems utilize recombinase interactions at chemically silent enhancer DNA sites to construct functional tetramers. Enhancer elements also function in transcription and replication systems. Knowledge gained from the study of Mu transposition should thus provide important insights into other complex biological reactions.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Texas Austin
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Jang, Sooin; Harshey, Rasika M (2015) Repair of transposable phage Mu DNA insertions begins only when the E.?coli replisome collides with the transpososome. Mol Microbiol 97:746-58
Harshey, Rasika M (2014) Transposable Phage Mu. Microbiol Spectr 2:
Choi, Wonyoung; Saha, Rudra P; Jang, Sooin et al. (2014) Controlling DNA degradation from a distance: a new role for the Mu transposition enhancer. Mol Microbiol 94:595-608
Choi, Wonyoung; Jang, Sooin; Harshey, Rasika M (2014) Mu transpososome and RecBCD nuclease collaborate in the repair of simple Mu insertions. Proc Natl Acad Sci U S A 111:14112-7
Saha, Rudra P; Lou, Zheng; Meng, Luke et al. (2013) Transposable prophage Mu is organized as a stable chromosomal domain of E. coli. PLoS Genet 9:e1003902
Jang, Sooin; Sandler, Steven J; Harshey, Rasika M (2012) Mu insertions are repaired by the double-strand break repair pathway of Escherichia coli. PLoS Genet 8:e1002642
Lee, Jaemin; Harshey, Rasika M (2012) Loss of FlhE in the flagellar Type III secretion system allows proton influx into Salmonella and Escherichia coli. Mol Microbiol 84:550-65
Harshey, Rasika M (2012) The Mu story: how a maverick phage moved the field forward. Mob DNA 3:21
Lazova, Milena D; Butler, Mitchell T; Shimizu, Thomas S et al. (2012) Salmonella chemoreceptors McpB and McpC mediate a repellent response to L-cystine: a potential mechanism to avoid oxidative conditions. Mol Microbiol 84:697-711
Ge, Jun; Lou, Zheng; Cui, Hong et al. (2011) Analysis of phage Mu DNA transposition by whole-genome Escherichia coli tiling arrays reveals a complex relationship to distribution of target selection protein B, transcription and chromosome architectural elements. J Biosci 36:587-601

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