Abstract
The site-specific recombinase integrase encoded by bacteriophage lambda 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.
Highlights
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]
The two carboxyl-terminal domains of Int, their interaction with core-type sites, and the mechanisms of DNA cleavage, strand exchange, and DNA ligation are all very similar to those of tyrosine recombinase family members Cre and Flp [6, 13]. Int is distinguished from these two family members by its aminoterminal domain and cognate arm-type binding sites
Arm-type Int Binding in the Excisive HJ Intermediate—To test predictions of the structure-based models, including the inference of N-domain binding to a non-canonical DNA site to the left of P2 in the excisive complexes and a non-canonical site to the left of P1 in the integrative complexes
Summary
Protein and DNA Preparation—Recombination proteins were purified as described previously (22, 34 –36). 2.5 nM attR substrates and 2.5 nM attL substrates were incubated with 125 nM Int, 10 nM IHF, 32 nM Xis, and 5 nM FIS (except for Fig. 4C, which was performed in the absence of FIS) in 25 mM Tris, pH 7.5, 6 mM spermidine, 5 mM EDTA, pH 8, 82 mM NaCl, 0.5 mg/ml BSA, and 2.5 mM dithiothreitol (DTT) for 2 h at 25 °C and terminated by the addition 4ϫ SDS loading buffer (1% SDS, 6% Ficoll) and loaded onto a 1.2% agarose gel and stained with ethidium bromide. For ternary complex gel shifts, 10 nM 32P-radiolabeled Holliday junction [40] and 400 nM concentrations of the indicated PЈ arm 40 bp oligonucleotide were incubated with 400 nM IntF (Y342F mutation) in 50 mM Tris, pH 7.4, 50 mM NaCl, 0.5 mg/ml BSA, 5 mM DTT, and 0.02 mg/ml herring sperm DNA for 60 min at 19 °C. Recovered DNA was ethanolprecipitated and resuspended in 100 l of 1 M piperidine, incubated at 90 °C for 30 min, dried via vacuum centrifugation, and loaded on a 10% polyacrylamide, 8 M urea sequencing gel
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