Donor transcription suppresses D-loops in cis and promotes genome stability

D-loops are DNA joint molecule intermediates central to DNA break repair by homologous recombination (HR). Priority rules between recombination and transcription at the donor locus have not been investigated. Here, using a controlled break induction system and physical detection of D-loops in S. cerevisiae , we show that donor transcription by RNA polymerase II acutely suppresses D-loops in cis , in an orientation-dependent manner. This inhibition does not rely on endogenous transcription factors, the RNA product, RNA:DNA hybrids, or previously characterized D-loop disruption factors. Transcription can be the major D-loop suppression pathway and inhibits the formation of repeat-mediated genome rearrangements. Transcription is therefore a negative regulator of HR at the D-loop level that promotes genome stability. These findings reveal the functional prioritization between two universal DNA-dependent processes.