Structure of a Holliday junction complex reveals mechanisms governing a highly regulated DNA transaction

Gurunathan Laxmikanthan,David Warren,Axel F Brilot,Wenjun Tong,Chen Xu,Lindsay Steele,Gregory D Van Duyne,Arthur Landy,Nicole Seah,Nikolaus Grigorieff

doi:10.7554/elife.14313

Gurunathan Laxmikanthan, David Warren + Show 8 more

Open Access

https://doi.org/10.7554/elife.14313

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Abstract

The molecular machinery responsible for DNA expression, recombination, and compaction has been difficult to visualize as functionally complete entities due to their combinatorial and structural complexity. We report here the structure of the intact functional assembly responsible for regulating and executing a site-specific DNA recombination reaction. The assembly is a 240-bp Holliday junction (HJ) bound specifically by 11 protein subunits. This higher-order complex is a key intermediate in the tightly regulated pathway for the excision of bacteriophage λ viral DNA out of the E. coli host chromosome, an extensively studied paradigmatic model system for the regulated rearrangement of DNA. Our results provide a structural basis for pre-existing data describing the excisive and integrative recombination pathways, and they help explain their regulation.

Highlights

The rearrangement of DNA, either by homologous or site-specific recombination and transposition, is a fundamental feature of evolution, genetic variation, and gene regulation; among such pathways, the integration and excision of bacteriophage l into and out of the E. coli chromosome is one of the most thoroughly characterized
The site-specific recombinase (Int) encoded by bacteriophage l is the archetypical member of the tyrosine recombinase family, whose members carry out such diverse functions as chromosome segregation, chromosome copy number control, gene expression, conjugative transposition, gene dissemination, and viral integration and excision (Craig et al, 2015; Jayaram et al, 2015; Landy, 2015)
The molecular details of this recombination are common to all reactions catalyzed by the large family of tyrosine recombinases, as first characterized for l Int, Cre, Flp, and XerC/D

Summary

Introduction

The rearrangement of DNA, either by homologous or site-specific recombination and transposition, is a fundamental feature of evolution, genetic variation, and gene regulation; among such pathways, the integration and excision of bacteriophage l into and out of the E. coli chromosome is one of the most thoroughly characterized. The Int subunits within a recombinogenic complex bind and bridge arm- and core-type DNA sites in patterns determined by the accessory DNA bending proteins IHF, Xis, and Fis. Differential occupancy of the 16 protein binding sites on the 240 bp att site generates two overlapping ensembles that differentiate the integrative and excisive recombination pathways ([Seah et al, 2014; Tong et al, 2014]; (see Figure 1). The molecular details of this recombination are common to all reactions catalyzed by the large family of tyrosine recombinases (except for the size of the O regions and the order of strand exchanges), as first characterized for l Int, Cre, Flp, and XerC/D (reviewed in [Van Duyne, 2015]) It proceeds in the absence of exogenous energy via the formation of high-energy covalent 3’phospho-tyrosine intermediates in the active site of each Int protein. The results provide a structural basis for understanding how these complex DNA recombination machines function and how they are so tightly regulated

Results and discussion

C Excisive Recombination

Materials and methods

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