Figure 2.

The C-terminal tail is required for efficient and accurate Holliday junction resolution. (a) Amount of total Holliday junction resolution product (top and bottom strand cleavages) by wild-type Int or W350ter. (b) Ratio of aberrant species to total Holliday junction resolution product at indicated protein concentrations of wild-type Int (open bars) and W350ter (filled bars). The aberrant species are likely single arm hairpins generated by uncoordinated Int cleavages, either during or after resolution.¹⁴,¹⁶ All Holliday junction resolution reactions ((a) and (b) and Figure 3(a)) were incubated at 20 °C and contain 120 nM integrase in assay volumes ranging from 10–100 μl with 50 mM Tris–HCl (pH 7.5), 50 mM NaCl, 0.5 mg/ml bovine serum albumin, 5 mM dithiothreitol, 10 nM ³²P-labeled Holliday junction substrate, and 240 nM P′ 1,2 arm oligonucleotides. Reactions were quenched with a SDS gel loading solution and separated on 7% (w/v) polyacrylamide Tris–glycine (pH 8.0), 0.1% (w/v) SDS gels. Gels were subsequently dried and analyzed with the Fuji BAS-2500 phosphorimager system. Wild-type and mutant Int proteins were prepared as described.²³ Int mutations were created via site-directed PCR mutagenesis with a Quikchange kit (Stratagene). Protein concentrations were determined by comparing the intensities of Coomasie-stained Int bands on an SDS-PAGE gel with those of a reference Int preparation quantified by amino acid composition (WM Keck Facility, Yale University). Preparation and sequences of synthetic Holliday junctions and P′ 1,2 oligonucleotides have been described.¹⁴