Figure 2. Interplay between Cas3 and RNaseHI affects plasmid copy number. (A) RNaseHI is needed for Cas3 to promote plasmid copy number. Experiments described in Figure 1A were repeated in MG1655 lacking RNaseHI (ΔrnhA) measuring yields of pUC19 as empty plasmid or expressing Cas3/Cas3^K320L, as indicated. Results are means of three independent tests in each case, with standard deviation error bars. (B) In this experiment, in addition to chromosomal rnhA, RNaseHI can also be overexpressed from the chromosome by arabinose induction from ParaBAD. Cells were transformed with pUC empty vector or pCas3 and then grown in either glucose (+ glu, to repress RNaseHI) or arabinose (+ ara, to induce RNaseHI) as indicated. The panel shows representative plasmid yield from 4 μl of pCas3 (uncut) extracted from cells grown in arabinose or glucose, electrophoresed in an ethidium bromide-stained agarose gel (0.8%).