The study of two different types of specialized recombination is proposed. Transpositional recombination, the integration of transposable DNA into a target site, will be investigated using two contrasting transposons as model systems. These are IS903, which generally transposes by a donor-destructive, non-replicative pathway, and GammaDelta which uses a replicative (cointegrate) pathway. Site specific recombination, the recombination between two specific sites by a reciprocal, conservative breakage reunion reaction, will be studied using the resolution of GammaDelta cointegrates by the transposon encoded TnpR protein, resolvase. The following specific projects are proposed. I Transpositional Recombination. 1) Purification and characterization of the transposase proteins of IS903 and GammaDelta, with transposition in vitro as the ultimate goal. 2) Analysis of the interaction of transposon ends and transposase (a) by mutational analysis of the transposon terminal inverted repeats and (b) by identification of DNA binding domains within the transposase protein by isolation of mutants of transposase that show altered sequence specificity. 3) Further analysis of the mechanism of deletion formation by IS903 and IS10. II Site-specific Recombination. 1) Mutational dissection of the cointegrate resolution process, (a) by developing assays for the various steps in recombination and (b) by isolating and characterizing mutants specifically defective in each step. 2) Detailed analysis of the molecular interactions between (a) the N-terminal domain of resolvase and the crossover site, and (b) the C-terminal domain and the resolvase-binding DNA segment. 3) Analysis of the apparent resolvase-induced bending of res DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028470-11
Application #
3275756
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1980-05-01
Project End
1991-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
11
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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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
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