Phage lambda Integrase (Int) is the prototype of a large family of tyrosine recombinases that performs site-specific recombination. The family includes bacterial, phage, conjugative transposon, integron and yeast plasmid enzymes. Many of these elements contribute to the virulence of pathogens and to the spread of antibiotic resistance. These proteins use the same mechanism of catalysis to cut and rejoin DNA strands as the structurally homologous type IB topoisomerases, of which the vaccinia virus topoisomerase and human topoisomerase I are the best studied examples. The central intermediate of tyrosine recombinases is a Holliday junction, a very transient pseudotetrasymmetric intermediate. One pair of integrase monomers (in a tetramer) performs one round of strand cleavage, exchange and religation to generate one Holliday junction isomer which must isomerize in order to activate the second pair of monomers for the second round of catalysis. The features of Int that control and stimulate this isomerization are still mysterious. We propose biochemical experiments to analyze the isomerization step and what controls its direction - this control is crucial to ensure that the reaction goes forward rather than backward, thus regulating the efficiency of recombination. For these experiments, we will make use of peptide inhibitors that we identified and characterized, whose advantage lies in trapping the Holliday junction intermediate in the recombination reaction. In order to increase the potential of these peptide inhibitors as tools, we propose to further characterize their interactions with Holliday junctions, both free and protein-bound. The new characterization will include structural studies of the inhibitors.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-IDM-H (02))
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Hagan, Ann A
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San Diego State University
Schools of Arts and Sciences
San Diego
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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
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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
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