The long-term objective of this program is to understand the molecular mechanism of conservative sitespecific recombination by the tyrosine recombinase family of enzymes and to apply this knowledge towards the design of improved tools for the manipulation of DNA molecules. The tyrosine recombinases catalyze strand exchanges between specific DNA sequences to accomplish a variety of important functions in biology, including the integration and excision of phage genomes and the maintenance of circular replicons. The Cre recombinase from bacteriophage P1 has emerged in the last decade to become both a powerful tool for the manipulation of genomes and a paradigm for structure and function in the tyrosine recombinase family. The impact of genomic engineering tools on human health is enormous, given the now widespread use of gene knockout and knock-in experiments, chromosome translocation experiments, and DNA rearrangements in molecular biology procedures. To the extent that an increased mechanistic understanding of these systems can be used to produce improved tools for molecular genetics, the proposed work will also have an important impact on human health.
The specific aims of this project are to (i) establish a structural and biochemical basis for understanding the directionality of the Cre-loxP recombination reaction, (ii) determine how Cre mutants and peptide inhibitors are able to support the production, but not the resolution, of the Holliday intermediate of the reaction, (iii) biochemically test specific functional hypotheses based on the Cre-DNA structural models, and (iv) establish a structural framework for understanding site-specific recombination in the more complex and tightly regulated Xer system.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM055041-09
Application #
7011228
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
1998-02-01
Project End
2008-01-31
Budget Start
2006-02-01
Budget End
2008-01-31
Support Year
9
Fiscal Year
2006
Total Cost
$252,394
Indirect Cost
Name
University of Pennsylvania
Department
Biochemistry
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Gibb, Bryan; Gupta, Kushol; Ghosh, Kaushik et al. (2010) Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res 38:5817-32
Ghosh, Kaushik; Guo, Feng; Van Duyne, Gregory D (2007) Synapsis of loxP sites by Cre recombinase. J Biol Chem 282:24004-16
Ghosh, Kaushik; Lau, Chi Kong; Guo, Feng et al. (2005) Peptide trapping of the Holliday junction intermediate in Cre-loxP site-specific recombination. J Biol Chem 280:8290-9
Guo, F; Gopaul, D N; Van Duyne, G D (1999) Asymmetric DNA bending in the Cre-loxP site-specific recombination synapse. Proc Natl Acad Sci U S A 96:7143-8
Gopaul, D N; Guo, F; Van Duyne, G D (1998) Structure of the Holliday junction intermediate in Cre-loxP site-specific recombination. EMBO J 17:4175-87