Reversible Inhibitors of λ Integrase-mediated Recombination Efficiently Trap Holliday Junction Intermediates and Form the Basis of a Novel Assay for Junction Resolution

Jeffrey L. Boldt, Clemencia Pinilla, Anca M. Segall

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

The bacteriophage λ 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.

Original languageEnglish (US)
Pages (from-to)3472-3483
Number of pages12
JournalJournal of Biological Chemistry
Volume279
Issue number5
DOIs
StatePublished - Jan 30 2004
Externally publishedYes

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