A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for λ Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination

Abstract

The site-specific recombinase integrase encoded by bacteriophage promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality. Integrase (Int)2 is a site-specific recombinase encoded by bacteriophage that promotes integration and excision of the viral chromosome into and out of the chromosome of the Escherichia coli host (1, 2). Int-mediated recombination between attP and attB on the and bacterial chromosomes, respectively, generates an integrated prophage bounded by attL and attR, which are also partners for the excisive recombination that reforms attP and attB (1, 3). Int is a member of a large family of tyrosine recombinases that execute site-specific recombination in the absence of high energy cofactors via a sequential pair of staggered (by seven base pairs, in the case of Int) single-strand exchanges that first generate and then resolve a four-way DNA junction known as a Holliday junction (HJ, see Fig. 1A) (4 -7). Each DNA strand cleavage involves nucleophilic attack by an active site tyrosine (Tyr-342) to form a high energy covalent 3Ј-phosphotyrosine bond. Attack of this intermediate by the 5Ј-hydroxyl of the swapped DNA strand forms one of the four ligated novel joints (8).Int is a well studied member of the subgroup of heterobivalent recombinases that bind and bridge two different families of DNA sequences (9). The amino-terminal domain of Int (N-domain, residues 1-63) binds with high affinity to arm-type Int binding sites (P1, P2, PЈ1, PЈ2, and PЈ3) that are distant from the core region where DNA strand exchange occurs (10) (Fig. 1A). Each of the core-type Int binding...

Nucleoprotein architectures regulating the directionality of viral integration and excision

“…In contrast, the NTD organization implied by the newly available core-arm bridging data for the excisive and integrative complexes requires considerable flexibility in the CB-NTD linkers and is incompatible with domain-swapped NTDs. Tellingly, the asymmetric complexes described here, unlike the symmetric arrangement in the smaller complexes designed for crystallization, are consistent with biotin-interference mapping of complete recombination reactions (44).…”

Teaching Cre to follow directions

“…Given that chimeric Cre has similar regulated directionality as -integrase and has the same N-domain binding site requirements, it is reasonable to conclude that tight coupling of the N-domain and CB domain is not required for regulated directionality. This idea is consistent with the presence of a flexible, protease-sensitive linker between domains in -integrase and helps to explain findings that the N-domain binding site requirements in the P and PЈ arms might require more flexibility than is implied by the compact N-domain tetramer observed in the crystal structures (9).…”

A Simple Topological Filter in a Eukaryotic Transposon as a Mechanism To Suppress Genome Instability

“…The conditions for two-dimensional gel electrophoresis were as previously described for Tn10 (6). Reverse gyrase, Tn10 transposase, lambda integrase, and integration host factor (IHF) were purified as described previously (3,7,15,21,28).…”