DNA rearrangements mediated by site-specific recombinases have important biological consequences. For example, site-specific recombination is responsible for integration and excision of phage genomes in bacteria, stable propagation of bacterial chromosomes during cell division, developmental control of gene expression in prokaryotes and copy number maintenance of extrachromosomal elements in yeasts. Understanding the mechanism of site-specific recombination will help us better understand DNA rearrangements that occur in living beings, prokaryotic as well as eukaryotic. Furthermore, the knowledge can be used to manipulate the reaction in a number of useful ways: to bring about targeted genetic alterations in bacteria, yeasts, plants, flies and mammalian systems. We have used the Flp site-specific recombinase from Saccharomyces cerevisiae as a simple model system for exploring the DNA-protein interactions and the chemical steps involved in the strand breakage and joining reactions. These studies have unveiled several global mechanistic features that characterize the recombination reactions carried out by the Integrase/Tyrosine family of proteins. We will now utilize the currently available molecular genetic, biochemical and structural information to further tackle some of the unsolved questions in the Flp system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035654-19
Application #
6712837
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Anderson, Richard A
Project Start
1990-01-01
Project End
2006-02-28
Budget Start
2004-03-01
Budget End
2005-02-28
Support Year
19
Fiscal Year
2004
Total Cost
$368,250
Indirect Cost
Name
University of Texas Austin
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
170230239
City
Austin
State
TX
Country
United States
Zip Code
78712
Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui et al. (2010) Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine. J Biol Chem 285:22976-85
Kachroo, Aashiq H; Ma, Chien-Hui; Rowley, Paul A et al. (2010) Restoration of catalytic functions in Cre recombinase mutants by electrostatic compensation between active site and DNA substrate. Nucleic Acids Res 38:6589-601
Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna et al. (2009) Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination. EMBO J 28:1745-56
Ma, Chien-Hui; Kachroo, Aashiq H; Macieszak, Anna et al. (2009) Reactions of Cre with methylphosphonate DNA: similarities and contrasts with Flp and vaccinia topoisomerase. PLoS One 4:e7248
Ma, Chien-Hui; Kwiatek, Agnieszka; Bolusani, Swetha et al. (2007) Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position. J Mol Biol 368:183-96
Du, Quan; Livshits, Alexei; Kwiatek, Agnieszka et al. (2007) Protein-induced local DNA bends regulate global topology of recombination products. J Mol Biol 368:170-82
Harshey, Rasika M; Jayaram, Makkuni (2006) The mu transpososome through a topological lens. Crit Rev Biochem Mol Biol 41:387-405
Bolusani, Swetha; Ma, Chien-Hui; Paek, Andrew et al. (2006) Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites. Nucleic Acids Res 34:5259-69
Yin, Zhiqi; Jayaram, Makkuni; Pathania, Shailja et al. (2005) The Mu transposase interwraps distant DNA sites within a functional transpososome in the absence of DNA supercoiling. J Biol Chem 280:6149-56
Konieczka, Jay H; Paek, Andrew; Jayaram, Makkuni et al. (2004) Recombination of hybrid target sites by binary combinations of Flp variants: mutations that foster interprotomer collaboration and enlarge substrate tolerance. J Mol Biol 339:365-78

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