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Summary: An important but still enigmatic function of DNA:RNA hybrids is their role in DNA double-strand break (DSB) repair. Here, we show that Sen1, the budding yeast ortholog of the human helicase Senataxin, is recruited at an HO endonuclease-induced DSB and limits the local accumulation of DNA:RNA hybrids. In the absence of Sen1, hybrid accumulation proximal to the DSB promotes increased binding of the Ku70-80 (KU) complex at the break site, mutagenic non-homologous end joining (NHEJ), micro-homology-mediated end joining (MMEJ), and chromosome translocations. We also show that homology-directed recombination (HDR) by gene conversion is mostly proficient in sen1 mutants after single DSB. However, in the absence of Sen1, DNA:RNA hybrids, Mre11, and Dna2 initiate resection through a non-canonical mechanism. We propose that this resection mechanism through local DNA:RNA hybrids acts as a backup to prime HDR when canonical pathways are altered, but at the expense of genome integrity.

The yeast RNA/DNA helicase Sen1, Senataxin in human, preserves the integrity of replication forks encountering transcription by removing RNA-DNA hybrids. Here we show that, in sen1 mutants, when a replication fork clashes head-on with transcription is arrested and, as a consequence, the progression of the sister fork moving in the opposite direction within the same replicon is also impaired. Therefore, sister forks remain coupled when one of the two forks is arrested by transcription, a fate different from that experienced by forks encountering Double Strand Breaks. We also show that dormant origins of replication are activated to ensure DNA synthesis in the proximity to the forks arrested by transcription. Dormant origin firing is not inhibited by the replication checkpoint, rather dormant origins are fired if they cannot be timely inactivated by passive replication. In sen1 mutants, the Mre11 and Mrc1–Ctf4 complexes protect the forks arrested by transcription from processing mediated by the Exo1 nuclease. Thus, a harmless head-on replication-transcription clash resolution requires the fine-tuning of origin firing and coordination among Sen1, Exo1, Mre11 and Mrc1–Ctf4 complexes.

RNA:DNA hybrids form when nascent transcripts anneal to the DNA template strand or any homologous DNA region. Co-transcriptional RNA:DNA hybrids, organized in R-loop structures together with the displaced non-transcribed strand, assist gene expression, DNA repair and other physiological cellular functions. A dark side of the matter is that RNA:DNA hybrids are also a cause of DNA damage and human diseases. In this review, we summarize recent advances in the understanding of the mechanisms by which the impairment of hybrid turnover promotes DNA damage and genome instability via the interference with DNA replication and DNA double-strand break repair. We also discuss how hybrids could contribute to cancer, neurodegeneration and susceptibility to viral infections, focusing on dysfunctions associated with the anti-R-loop helicase Senataxin.