Figure 5: R-loops are not needed for mutagenesis when DSBs are caused by I-SceI.

Overproduction of RNase HI (reduction in R-loops) does not decrease mutagenesis when I-SceI-generated DSBs are provided. (a) The RNase HI-overproducing strain (PJH1239) (♦) shows about half as much Lac⁺ mutation as the strain carrying the control plasmid (PJH1237) (). (b) The DSB-producing strain (carrying the chromosomal inducible I-SceI enzyme and I-SceI cutsite near lac) that over-produces RNase HI (PJH1321) (♦) shows no reduction of Lac⁺ mutants compared with the isogenic strain carrying the control plasmid (PJH1319) (), indicating that R-loops are not necessary for mutation when DSBs are provided. This result holds when both I-SceI enzyme and cutsite are present and not in strains with the enzyme only (a). (c) Strains experiencing double-strand cutting by I-SceI show reduced viability. However, the viability decrease is the same for cells with the rnhA plasmid as for those with the control plasmid making quantitative comparisons meaningful. Relative viability was determined per²¹. Values in (a,b) have not been corrected for declining viability. Error bars represent one s.e.m. of four parallel cultures. Each experiment was performed three times with comparable results.