Lambda site-specific recombination (SSR), like all recombination reactions, requires the synapsis or juxtaposition of two DNA substrates by recombination proteins and their accessory factors into a productive arrangement. The protein-DNA complexes assembled contain several different proteins serving both catalytic and/or architectural roles (e.g., bending DNA). Lambda SSR is now known to comprise four distinct pathways. The integration and excision pathways, used respectively to join and separate the lambda and E. coli genomes, are unidirectional, irreversible reactions where the products differ from the substrates. These two reactions differ in two fundamental ways: they contain different DNA partners (attB and attP vs attL and attR) and they have different protein requirements (int vs int+xis). The two other pathways, between two attL or two attP sites, promoted by Int are bidirectional and the products look like the substrates. Therefore, the four pathways are distinguished both by the structure of the DNA substrates and by the proteins which help Int mediate the reaction: the chemical steps of cleaving and resealing the DNA take place in four distinct protein-DNA environments. This application focuses on examining how protein-DNA and protein-protein interactions in the four pathways determine differences among the pathways, by separating features of Int that are required for chemical catalysis from features that influence directionality. To that end, four specific aims are delineated: 1- A fine-structure analysis of interactions between Int and the regions of strand exchange will be carried out for a second synaptic recombination intermediate to compare these interactions between two bidirectional pathways of recombination. 2- An in-depth in vitro analysis of existing Int variants, which are able to recombine through some pathways but not others, will be performed to determine which features are required for all recombination pathways and which are required for individual pathways. This analysis will determine similarities and differences among the pathways. 3- The nature and role of protein-protein interactions within a recombination complex will be investigated for different pathways. Specifically, the contacts between Int monomers will be determined. 4- A new tool, based on a ligation-defective Int mutant, will be developed to study transient synaptic complexes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM052847-02S1
Application #
2698242
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1996-08-01
Project End
2000-07-31
Budget Start
1997-11-01
Budget End
1998-07-31
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
San Diego State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
Rideout, Marc C; Naili, Ilham; Boldt, Jeffrey L et al. (2013) wrwyrggrywrw is a single-chain functional analog of the Holliday junction-binding homodimer, (wrwycr)2. Peptides 40:112-22
Orchard, Samantha S; Rostron, Jason E; Segall, Anca M (2012) Escherichia coli enterobactin synthesis and uptake mutants are hypersensitive to an antimicrobial peptide that limits the availability of iron in addition to blocking Holliday junction resolution. Microbiology 158:547-59
Rideout, Marc C; Boldt, Jeffrey L; Vahi-Ferguson, Gabriel et al. (2011) Potent antimicrobial small molecules screened as inhibitors of tyrosine recombinases and Holliday junction-resolving enzymes. Mol Divers 15:989-1005
Ranjit, Dev K; Rideout, Marc C; Nefzi, Adel et al. (2010) Small molecule functional analogs of peptides that inhibit lambda site-specific recombination and bind Holliday junctions. Bioorg Med Chem Lett 20:4531-4
Gunderson, Carl W; Boldt, Jeffrey L; Authement, R Nathan et al. (2009) Peptide wrwycr inhibits the excision of several prophages and traps holliday junctions inside bacteria. J Bacteriol 191:2169-76
Fujimoto, David F; Higginbotham, Robin H; Sterba, Kristen M et al. (2009) Staphylococcus aureus SarA is a regulatory protein responsive to redox and pH that can support bacteriophage lambda integrase-mediated excision/recombination. Mol Microbiol 74:1445-58
Rajeev, Lara; Segall, Anca; Gardner, Jeffrey (2007) The bacteroides NBU1 integrase performs a homology-independent strand exchange to form a holliday junction intermediate. J Biol Chem 282:31228-37
Fujimoto, David F; Pinilla, Clemencia; Segall, Anca M (2006) New peptide inhibitors of type IB topoisomerases: similarities and differences vis-a-vis inhibitors of tyrosine recombinases. J Mol Biol 363:891-907
Gunderson, Carl W; Segall, Anca M (2006) DNA repair, a novel antibacterial target: Holliday junction-trapping peptides induce DNA damage and chromosome segregation defects. Mol Microbiol 59:1129-48
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

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