Fig 2.

The Rep helicase domain binds DNA when the linker region is appended. Binding was assessed on a Superdex 200 column based on comigration of protein and DNA during SEC after Rep221-489 (left) or Rep198-489 (right) at 82 μM (2.5 and 2.8 mg/ml, respectively) was mixed with a 6:1.2 molar ratio of the indicated DNA oligonucleotides and then dialyzed overnight in binding buffer (see Materials and Methods). The resulting chromatograms are shown in panels B to E. Protein alone similarly dialyzed is shown in panel A. Note that Rep198-489 is markedly less soluble than Rep221-489 in binding buffer. The A₂₆₀ is shown in red, the A₂₈₀ is shown in black, and the elution position of blue dextran (corresponding to the column void) is marked. The complex at ∼22.5 min that is formed with Rep198-489 is indicated. (F) SDS-PAGE of Rep198-489 fractions collected during chromatography; the indicated fraction number corresponds to the elution time in minutes. The two molecular weight (MW) markers on the left of each gel (Mark12; Invitrogen) represent M_r values of 36,000 (top) and 31,000 (bottom). (G) Standard curve used to determine the apparent molecular masses (MM) of Rep198-489/DNA complexes. The elution positions on a Superdex 200 column of the molecular mass standards (•) thyroglobulin (thy; 669 kDa), ferritin (fer; 443 kDa), catalase (cat; 232 kDa), aldolase (ald; 158 kDa), and bovine serum albumin (BSA; 67 kDa) are plotted on a semilog plot as log(MM) versus V_e/V_o, where V_e is the elution volume and V_o is the void volume elution time. The curve yields an apparent molecular mass of ∼385 kDa for the Rep198-489 complexes eluting at ∼22.5 min (red circle).