Figure 3. Cas3 provokes formation of plasmid concatemers. (A) Lanes 1–4 show ethidium bromide-stained agarose electrophoresis of uncut pCas3 or pCas3^K320L from two independent plasmid extractions, to illustrate typical pCas3 concatemer formation including high molecular mass species that remain in gel wells (lane 3). Restriction digestion of uncut pCas3 converted multimeric plasmid (lane 5) into a single product consistent with linear pCas3 (5.4 kbp, lane 6). The DNA size marker is Fermentas GeneRuler. (B) Agarose gel showing concatemer (C) formation of pUC19 extracted from ΔrnhA cells (lane 2) compared with rnhA⁺ cells (lane 1). This effect is typical of cells lacking RNaseHI, as reported previously.³⁰ IPTG induced expression of chromosomal Cas3 from PlacUV5 further increases concatemer formation of pUC19 (lane 3). All DNA species from PlacUV5-Cas3 were converted to single band of linear pUC19 after restriction digestion (lane 4).