Reversible inhibitors of lambda integrase-mediated recombination efficiently trap Holliday junction intermediates and form the basis of a novel assay for junction resolution.
The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.
Boldt
Jeffrey L
JL
Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California 92182-4614, USA.
Pinilla
Clemencia
C
Segall
Anca M
AM
D010582:Bacteriophage lambda / Q000201:enzymology; D002384:Catalysis; D020650:Combinatorial Chemistry Techniques; D004247:DNA / Q000737:chemistry; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D019429:Integrase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D019426:Integrases / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D013329:Structure-Activity Relationship; D014616:Vaccinia virus / Q000201:enzymology; D014764:Viral Proteins / Q000378:metabolism
D004220:Disulfides; D019429:Integrase Inhibitors; D008301:Maleimides; D010455:Peptides; D014764:Viral Proteins; C075594:1,6-bismaleimidohexane; D004247:DNA; C045073:Cre recombinase; D019426:Integrases; D004262:DNA Restriction Enzymes; D004264:DNA Topoisomerases, Type I; D004229:Dithiothreitol