The overall goal of this project is to understand, in as much detail as possible, the mechanisms of two different kinds of specialized recombination associated with transposable DNA elements: site-specific recombination and transpositional recombination. The model system for site-specific recombination - a breakage-exchange-reunion reaction between two specific sites - is the resolution of cointegrates (the product of transpositional recombination by the gamma delta transposon) by the gamma delta resolvase, prototype of the serine recombinases. Transpositional recombination will be studied using a transposon of the D,D(35)E superfamily, Tn552. Site-specific recombination mediated by the gamma delta resolvase. A primary goal is to elucidate the architecture of the synaptic complex (containing two 120 bp ressites and 6 dimers of resolvase) within which recombination occurs. We have formulated a new structural model for this complex based on crystallography of the resolvase dimer and a detailed knowledge of interdimer interactions. A variety of approaches, particularly fluorescence resonance energy transfer (FRET), will be used both to test predictions of this (and an opposing) model, and also to probe for large rearrangements of the resolvase subunits or domains that accompany the activation of catalytic functions and the process of strand exchange. An investigation of another related serine recombinase, the transposase of the Helicobactor pylori element, IS60 7, will also be initiated. Transpositional recombination by Tn552. We have developed an efficient in vitro strand transfer reaction for Tn552, using the TnpA transposase and a transposon substrate with pre-cleaved ends. We intend to focus on the role of the accessory transposition protein, TnpB in activation of TnpA (allowing it to cleave uncleaved transposon ends) and in transposition immunity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028470-25
Application #
6887409
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Portnoy, Matthew
Project Start
1980-05-01
Project End
2008-04-30
Budget Start
2005-05-01
Budget End
2008-04-30
Support Year
25
Fiscal Year
2005
Total Cost
$408,750
Indirect Cost
Name
Yale University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
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
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
Lu, Jin-Ying; Lin, Yu-Yi; Qian, Jiang et al. (2008) Functional dissection of a HECT ubiquitin E3 ligase. Mol Cell Proteomics 7:35-45
Tao, Sheng-Ce; Li, Yu; Zhou, Jiangbing et al. (2008) Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers. Glycobiology 18:761-9
Gehman, John D; Cocco, Melanie J; Grindley, Nigel D F (2008) Chemical shift mapping of gammadelta resolvase dimer and activated tetramer: mechanistic implications for DNA strand exchange. Biochim Biophys Acta 1784:2086-92
Kamtekar, Satwik; Ho, Roger S; Cocco, Melanie J et al. (2006) Implications of structures of synaptic tetramers of gamma delta resolvase for the mechanism of recombination. Proc Natl Acad Sci U S A 103:10642-7
Li, Weikai; Kamtekar, Satwik; Xiong, Yong et al. (2005) Structure of a synaptic gammadelta resolvase tetramer covalently linked to two cleaved DNAs. Science 309:1210-5
Lewis, Leslie A; Cylin, Edruge; Lee, Ho Kyung et al. (2004) The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway. J Bacteriol 186:858-65
Leschziner, Andres E; Grindley, Nigel D F (2003) The architecture of the gammadelta resolvase crossover site synaptic complex revealed by using constrained DNA substrates. Mol Cell 12:775-81
Kirby, Carolyn; Waring, Al; Griffin, Thomas J et al. (2002) Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue. Mol Microbiol 43:173-86

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