Figure 7.

RecF over-expression increases damage and ssDNA formation on plasmid DNA. The effect of RecF ATPase over-expression on plasmid integrity was determined by quantitative electrophoresis and electron microscopy. Cells were grown for 16 h in presence or absence of 10% arabinose. (A) Plasmids isolated from cells as mentioned. Purified plasmid DNA (250 ng) was loaded onto two identical 1% agarose gels. After electrophoresis DNA was EtBr or SYBR Gold stained. The upper panel represent the gel images of representative experiments and lower panel represent the average of the raw intensity signal detected for the major band for a biological triplicate ±SD. The P values of significant differences between staining methods are indicated in grey and differences relative to the wt strain in the same conditions are indicated respectively in orange and pink for EtBr and SYBR Gold. (B) Electron microscopy images of pBR322 purified DNA using the cytochrome C spreading method. The cytochrome C spreading allowed the differentiation between the dsDNA (black arrow) and the ssDNA (white arrow). RecF over-expression led to an apparent increase in observed ssDNA, concomitant to an almost complete loss of the circular dsDNA in the plasmid preps.