Figure 3
Production of wild-type and mutant RecA proteins. An improved SUMO fusion protein expression system was used to produce authentic RecA protein. (A) Purified RecA protein on a 12% SDS-PAGE gel stained with Coomassie Blue. (B) Nuclease activity assay. Purified RecA protein (1 Ī¼M) or exonuclease I (20 units; New England Biolabs) was incubated with 5'-end 32P-labeled P1656 ssDNA (50 nucleotides, 3 Ī¼M), respectively. The reaction mixtures were treated with proteinase K and then electrophoresed on 20% native acrylamide gels. Gels were visualized on a phosphorimager, with overexposure to confirm that the purified RecA proteins exhibited no detectable nuclease activity. (C) Purified RecA proteins are capable of polymerization into nucleoprotein helical filaments. Negative-staining electron microscopy images show various RecA proteins with a Ī¦X174 dsDNA substrate in the presence of ATPĪ³S. Scale bars (in black) are 100 nm.