Peptide Trapping of the Holliday Junction Intermediate in Cre-loxP Site-specific Recombination

Kaushik Ghosh,Chi Kong Lau,Feng Guo,Anca M Segall,Gregory D Van Duyne

doi:10.1074/jbc.m411668200

Abstract

Cre recombinase is a prototypical member of the tyrosine recombinase family of site-specific recombinases. Members of this family of enzymes catalyze recombination between specific DNA sequences by cleaving and exchanging one pair of strands between the two substrate sites to form a 4-way Holliday junction (HJ) intermediate and then resolve the HJ intermediate to recombinant products by a second round of strand exchanges. Recently, hexapeptide inhibitors have been described that are capable of blocking the second strand exchange step in the tyrosine recombinase recombination pathway, leading to an accumulation of the HJ intermediate. These peptides are active in the lambda-integrase, Cre recombinase, and Flp recombinase systems and are potentially important tools for both in vitro mechanistic studies and as in vivo probes of cellular function. Here we present biochemical and crystallographic data that support a model where the peptide inhibitor binds in the center of the recombinase-bound DNA junction and interacts with solvent-exposed bases near the junction branch point. Peptide binding induces large conformational changes in the DNA strands of the HJ intermediate, which affect the active site geometries in the recombinase subunits.

Highlights

The tyrosine recombinases include the well-studied bacteriophage ␭-integrase, Cre recombinase from bacteriophage P1, Flp recombinase from the Saccharomyces cerevisiae 2-micron circle, the bacterial XerC/D recombinases, and well over 100 other proteins identified among bacteria and yeast [1, 2]
Specific Interaction of WKHYNY with the Cre-loxPHJ Intermediate—To determine whether the WKHYNY peptide binds to the Holliday junction (HJ) intermediate or if it binds to other intermediates and components of the Cre-loxP system, we used a fluorescence-based assay that can be performed in solution under equilibrium conditions
The biochemical and structural data presented here strongly support a model in which peptide inhibitor binds at the center of the Cre-bound HJ intermediate

Summary

Introduction

The tyrosine recombinases (formerly referred to as the ␭-integrase family) include the well-studied bacteriophage ␭-integrase, Cre recombinase from bacteriophage P1, Flp recombinase from the Saccharomyces cerevisiae 2-micron circle, the bacterial XerC/D recombinases, and well over 100 other proteins identified among bacteria and yeast [1, 2]. By iterative screening of mixed pools of synthetic peptides for their ability to block specific recombination steps, several potent inhibitors have been identified for both the initial synaptic complex, where the initial strand exchange is blocked, and for the HJ intermediate, where resolution of the junction in either direction is blocked. In addition to their utility as mechanistic probes to dissect the complex mechanism of ␭-integrase sitespecific recombination, such inhibitors could have more general utility in other biological pathways, those that generate HJ intermediates [16]

Methods

Results

Conclusion