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. 2006 May 30;4(6):e186. doi: 10.1371/journal.pbio.0040186

Figure 7. Substrate-Dependent Interaction of Bxb1 gpInt and gp47.

Figure 7

(A and B) Binding of gpInt and gp47 to attP/ attB/ attL/ attR was monitored by native gel electrophoresis. Radiolabeled DNA fragments (˜300 bp) were incubated with either gpInt (0.072 μM) alone or gpInt with increasing concentrations of gp47 (0.45, 0.89, 1.78, and 3.56 μM). The positions of DNA–gpInt complexes (cmplx I) as well as tertiary complexes containing DNA, gpInt, and gp47 (cmplx II) are shown.

(C and D) The presence of gp47 in the tertiary complexes shown in (A) was determined by the ability of α-His antibodies to supershift complexes observed by native gel electrophoresis. α-His antibodies were either added to reactions containing DNA, gpInt, and gp47 (indicated as lane 1), or first preincubated with gp47 for 30 min and then added to reactions containing DNA and gpInt (lane 2). The protein–DNA complexes were separated from free DNA on a 5% native PAGE. The positions of the tertiary complexes of gp47, gpInt, and DNA as well as the antibody supershifted complexes are indicated.

(E and F) Bxb1 gp47 is required for trapping a synaptic complex in excision. A suicide substrate version of attL DNA (5′ radiolabeled at both ends) was used that has a nick on the top strand positioned four bases to the 5′ side of the scissile bond. Bxb1 gpInt (72 nM) binds normally to this substrate to form Complex I (cmplx I), but when attR partner DNA (200 bp) and gp47 (3.56 μM) is added, no recombinant products are released. Instead, a prominent slow-moving complex is observed that absolutely requires Bxb1 gp47 for its formation. We have identified this as a synaptic complex using 2D-PAGE (F). In brief, a vertical gel slice was removed from the last lane in panel E, incubated with proteinase K and SDS, and then electrophoresed through a second dimension. Approximately 50% of the radiolabeled DNAs in this complex correspond to attP recombinant product and 50% correspond to a cleaved half-site. The bottom of the gel slice containing unbound attL DNA was removed prior to the second dimension of electrophoresis. Further details on the characterization of these suicide substrates will be described in future publications.