Figure 1. A new genetic assay to detect recombinogenic DNA:RNA hybrids in trans.

(A) DSBs induced in between direct repeats by DNA:RNA hybrids putatively formed with RNA produced in trans would be repaired by Rad51-independent Single-Strand Annealing (SSA) causing the deletion of one of the repeats. A DSB is depicted for simplicity, but other recombinogenic lesions such as nicks or ssDNA gaps cannot be ruled out. (B) Schematic representation of the recombination assay to study the recombinogenic potential RNA produced by transcription (Trx) in cis or in trans. Four combinations were studied: i) no transcription, with GL-LacZ construct turned transcriptionally off (2% glucose) and an empty plasmid; ii) transcription in trans, with GL-LacZ construct turned transcriptionally off (2% glucose) and the tet_p:LacZ construct; iii) transcription in cis, with GL-LacZ construct turned transcriptionally on (2% galactose) and an empty plasmid; and iv) transcription in cis and in trans, with GL-LacZ construct turned transcriptionally on (2% galactose) and the tet_p:LacZ construct.

Figure 1—figure supplement 1. LacZ expression levels in the GL-LacZ and tet_p:LacZ constructs.

Relative RNA levels at the LacZ gene from GL-LacZ and tet_p:LacZ constructs when transcription was turned either off (Trx -) or on (Trx +) in WT (W303), hpr1Δ (U678.4C) and rnh1Δ rnh201Δ (HRN2.10C) strains transformed with either pRS314-GL-LacZ or pCM179. Transcription at the GL-LacZ construct was turned on or off by growth in media with 2% galactose or 2% glucose, respectively. Transcription at the tet_p:LacZ construct was turned on or off by growth in media without or with 5 μg/mL doxycycline, respectively. Average and SEM of three independent experiments are shown.