Figure 6.

BcgI reactions on plasmids with one or two sites. The plasmids pRMS01 and pRMS02 (that have, respectively, one or two sites for BcgI) were isolated from dam⁺ and dam⁻ strains of E. coli as SC DNA; the DNA from the dam⁺ strain has HM BcgI sites and pRMS01 and pRMS02 are noted as 1H and 2H, respectively; the plasmids from the dam⁻ strain have UM BcgI sites and are noted as 1U and 2U. The plasmids were used for DNA cleavage (a) and DNA methylation (b) reactions by the BcgI RM protein. (a) Cleavage reactions contained, in buffer R* at 37°C, BcgI protein (20 nM) and one of the following plasmids (5 nM) as indicated above the gel image; 1H (one HM site); 1U (one UM site); 2H (two HM sites) and 2U (two UM sites). Samples were withdrawn at the times indicated above the gel (T, min) and analysed by electrophoresis though agarose to separate the SC, open circle (OC), dimeric and linear (LIN) forms of each plasmid and, for those with two sites, the products cut at both sites (L1 and L2). (b) Methylation reactions in buffer M at 37°C contained 50 nM BcgI protein and 100 nM plasmid DNA from the dam⁺ strain; either 1H, white circles; or 2H, black circles. Samples were withdrawn from the reaction at the times indicated and the transfer of the ³H label from the SAM to the DNA measured as in the ‘Materials and Methods’ section. The dpm readings were normalized to the number of BcgI sites in the DNA. Each data point is the mean of three repeats (with standard deviations shown) and the line drawn through each set is the best fit to an exponential.