FIG. 5.

Gel retardation assay showing that the binding between the Rep₂₀₀₉ protein and ori₂₀₀₉ is the result of a specific interaction. Gel retardation was performed as described in Materials and Methods. An 0.3-ng amount of ³²P-labeled ori₂₀₀₉ DNA fragment (160-bp Per-conferring region) was used as a probe. Lanes: 1, probe only; 2, probe plus cell extract (2.5 μg of total protein) from L. lactis NZ9800 harboring pNZ8048 (negative control); 3 to 15, probe plus cell extract (2.5 μg of total protein) from L. lactis NZ9800 containing overexpressed Rep₂₀₀₉ protein (approximate Rep₂₀₀₉ protein molecular mass, 29 kDa). Lanes 4 to 9 represent binding assays performed with the labelled ori₂₀₀₉ probe, cell extract, and 1.2, 2.4, 4.8, 9.6, 19.2, and 38.4 ng of unlabelled ori₂₀₀₉ DNA fragment, respectively. Lanes 10 to 15 represent binding assays performed with the labelled ori₂₀₀₉ probe, cell extract, and 1.2, 2.4, 4.8, 9.6, 19.2, and 38.4 ng of an unlabelled nonspecific DNA fragment of approximately the same size as the labelled DNA fragment, respectively.