Figure 1
Effects of UV irradiation on CRP-DNA complexes and isolated CRP assessed by gel mobility shift assays (A,B) and SDS-PAGE (C,D). Panel A: Gel mobility shift assay of CRP-cAMP complexes (50 µM cAMP) incubated in the dark, or exposed to UV (20 min exposure, λmax 310 nm, 30 nm band width, 6 W m−2, normoxia), before addition to 32P-labeled DNA in CRP binding buffer (see Materials and methods). DTT (1 mM), NaN3 (100 mM) and mannitol (100 mM) were present as indicated. Lane 1: no CRP; lanes 2–4: CRP at 2.5, 5 and 10 nM respectively with no UV exposure; lane 5–7: as lanes 2–4, respectively, but with UV-exposed CRP; lanes 8–10: as lanes 5–7, respectively, but with DTT; lanes 11–13: as lanes 5–7, respectively, but with NaN3; lanes 14–16: as lanes 5–7, respectively, but with mannitol. Panel B. Pre-formed CRP-cAMP (50 µM)-DNA complexes were incubated in the dark, or exposed to UV, in the absence or presence of additives (as panel A). Lane 1: no CRP; lanes 2–5: CRP at 1.25, 2.5, 5 and 10 nM, respectively, with no UV exposure; lane 6–8: UV-exposed CRP complex at 2.5, 5 and 10 nM, respectively; lanes 9–11: as lanes 6–8, but with DTT; lanes 12–14: as lanes 6–8, but with NaN3; lanes 15–17: as lanes 6–8, but with mannitol. Panel C. SDS-PAGE of isolated CRP (5 μM)-cAMP (50 µM) complex incubated in the dark (lane 1), or exposed to UV (as panel A) for 5 (lane 2) or 20 min (lane 3). Samples were run under reducing conditions, before visualization with InstantBlue staining. Reactions were carried out in buffer containing: 10 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 55 µg mL−1 bovine serum albumin, 0.05% NP-40 and 2 µg mL−1 calf thymus DNA. Panel D. SDS-PAGE analysis of CRP (5 μM)-cAMP (50 µM) complex incubated in the dark, or exposed to UV (as panel A), in the absence or presence of plasmid DNA. Lane 1: CRP alone, no UV; lane 2: CRP with 1 μg plasmid DNA, no UV; lane 3: CRP alone with UV; lanes 4–6: UV-exposed CRP with 0.2, 1 and 3 μg plasmid DNA, respectively. Reactions were carried out in buffer containing:10 mM Tris–HCl (pH 8.0), 50 mM KCl, 2.5 mM MgCl2, 1 mM EDTA, 55 µg mL−1 bovine serum albumin and 0.05% NP-40.
