Figure 5.

Cyclin A proteolytic activity is present in DNA preparation and in bacterial lysates. (A) Inhibition of cleavage of cyclin A by benzamidine. GST-cyclin A(CΔ114) was incubated with cyclin B(RL) in the presence of buffer (lane 2), E64 (lane 3), benzamidine (lane 4), or soybean trypsin inhibitor (lane 5) at 37°C for 120 min. Cyclin A cleavage was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards in kDa are indicated on the left. (B) Cyclin B in pET21d DNA (lanes 4–9) were boiled for 5 min (lane 5), subjected to ethanol precipitation (lane 8), or treated with benzimidine (lane 6), DNase (lane 7), or proteinase K (lane 9). The proteinase K was subsequently inactivated by phenol/chloroform extraction. The samples were then incubated with purified GST-cyclin A(CΔ114) for the indicated time. Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining (Upper). Molecular size standards (lane 1) in kDa are indicated on the left. DNA in the samples was visualized by agarose gel electrophoresis and ethidium bromide staining (Lower). (C) GST-cyclin A(CΔ114) was incubated with buffer (lanes 2 and 3), or DH5α lysates (lanes 4–9) in the presence of ZnCl₂ (0.1 mM, lane 5; 1 mM, lane 6) or CuCl₂ (0.1 mM, lane 7; 1 mM, lane 8; 10 mM, lane 9). Cleavage of cyclin A was detected by SDS/PAGE and Coomassie blue staining. Molecular size standards (lane 1) in kDa are indicated on the left.