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. Author manuscript; available in PMC: 2011 Jun 15.
Published in final edited form as: Arch Biochem Biophys. 2010 Apr 20;498(2):89–94. doi: 10.1016/j.abb.2010.04.011

Figure 3. Formation of a stable Metnase-hPso4-DNA complex does not require Metnase’s DNA binding activity.

Figure 3

Figure 3

Figure 3

A. SDS-PAGE of immunoaffinity purified Flag-Metnase (lane 1) and Flag-hPso4 (lane 2) used in this study. Lane M represents protein markers. B & C. Formation of a stable Metnase-hPso4 complex with TIR and non-TIR DNA. Reaction mixtures (20 ul) containing fixed amount of Metnase (0 or 1.0 pmol) were incubated with varying amounts of hPso4 (0.25, 0.5, 1.0, and 2.0 pmol) for 15 min prior to addition of 400 fmol of 5′-32P-labeled TIR DNA (panel B) or non-TIR DNA (MAR3M, panel C). Following 15 min incubation at 25°C, the protein-DNA complexes were analyzed by 5% native PAGE in the presence of 1X TBE. For quantification, individual bands were excised from dried gel and measured for radioactivity. D. A Metnase mutant (R432A) lacking DNA binding activity form a stable complex with hPso4 and dsDNA is independent of Metnase’s DNA binding activity. Reaction mixtures (20 ul) containing indicated amounts of R432A lacking TIR-specific DNA binding activity and/or hPso4 were mixed with 200 fmol of 5′-32P-labeled TIR and incubated for 15 min at 25°C, and analyzed by 5% native PAGE.