Tuberculosis is the leading cause of death from a single infectious agent, i.e. Mycobacterium tuberculosis. Development of genetic tools for understanding mycobacterial biology has been advanced through the study of their viruses, the mycobacteriophages. Several well-characterized mycobacteriophages form lysogens in which the phage DNA is integrated into the host chromosome and these integration systems have proven useful both for constructing site-specific integration-proficient vectors but also for elucidating the mechanism and control of site-specific recombination. Several newly characterized mycobacteriophages integrate and excise their DNA using unusual serine-integrases, which contain a catalytic motif similar to those in transposon resolvases and DNA-invertases. A similar protein is involved in the mobility of the prophage-like element, phiRvl, in M. tuberculosis. However, little is known about how this class of enzymes catalyzes integrative recombination between their attP and attB sites, or how this same protein catalyzes recombination between attL and attR for excision. Since attP and attB are different, an interesting biochemical and structural problem arises as to how these serine-integrases choose the correct site pairs for productive recombination. Defined in vitro reactions have been established for both phiRvl and phage Bxbl integration and are simple, requiring just the correct DNA partners, purified integrase and a simple buffer. There is no requirement for either DNA supercoiling, additional proteins or high-energy cofactors. An in vitro assay has also been established for phiRvl excision, which requires a phiRvl-encoded recombination directionality factor (RDF). The mechanism and control of these reactions will be determined through analysis of the requirements at the attachment sites, the nature of the interaction between the integrase and RDF proteins and DNA, and mutational dissection of the integrase proteins. These studies will provide important insights into how phage integration and excision operate and how they can be harnessed as genetical tools for understanding M. tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059114-03
Application #
7010678
Study Section
Special Emphasis Panel (ZRG1-MBC-2 (01))
Program Officer
Sizemore, Christine F
Project Start
2004-02-15
Project End
2009-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
3
Fiscal Year
2006
Total Cost
$352,806
Indirect Cost
Name
University of Pittsburgh
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
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Singh, Shweta; Rockenbach, Kate; Dedrick, Rebekah M et al. (2014) Cross-talk between diverse serine integrases. J Mol Biol 426:318-31
Broussard, Gregory W; Hatfull, Graham F (2013) Evolution of genetic switch complexity. Bacteriophage 3:e24186
Singh, Shweta; Ghosh, Pallavi; Hatfull, Graham F (2013) Attachment site selection and identity in Bxb1 serine integrase-mediated site-specific recombination. PLoS Genet 9:e1003490
Broussard, Gregory W; Oldfield, Lauren M; Villanueva, Valerie M et al. (2013) Integration-dependent bacteriophage immunity provides insights into the evolution of genetic switches. Mol Cell 49:237-48
Bai, Hua; Kath, James E; Zorgiebel, Felix Manuel et al. (2012) Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end. Proc Natl Acad Sci U S A 109:16546-51
Savinov, Andrew; Pan, James; Ghosh, Pallavi et al. (2012) The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication. Gene 495:42-8
Bai, Hua; Sun, Mingxuan; Ghosh, Pallavi et al. (2011) Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase. Proc Natl Acad Sci U S A 108:7419-24
Ghosh, Pallavi; Bibb, Lori A; Hatfull, Graham F (2008) Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading. Proc Natl Acad Sci U S A 105:3238-43
Ghosh, Pallavi; Wasil, Laura R; Hatfull, Graham F (2006) Control of phage Bxb1 excision by a novel recombination directionality factor. PLoS Biol 4:e186

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