Dormant origins and fork protection mechanisms rescue sister forks arrested by transcription

Abstract

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.

Sen1 Is Recruited to Replication Forks via Ctf4 and Mrc1 and Promotes Genome Stability

“…It has been previously proposed (using the sen1-1 allele or Sen1 depletion) that Sen1's presence at RFs is required to quickly remove the R-loops accumulating and interfering with RF progression (Alzu et al, 2012;Brambati et al, 2018;Mischo et al, 2011). In our experimental setting, however, loss of Sen1 from RFs did not show increases in DNA:RNA hybrids or dramatic defects in RF progression (Figures 4G and 5C).…”

“…Because of these defects, the viability of sen1-1 cells depends on several repair factors (Mischo et al, 2011;Alzu et al, 2012). Moreover, depletion of Sen1 leads to slow DNA replication and the accumulation of abnormal structures on 2D gels (Alzu et al, 2012;Brambati et al, 2018). Given its relatively low abundance and processivity (Mischo et al, 2018;Han et al, 2017), Sen1 needs to be recruited at, or close to, sites where it can enact its biological function.…”

Senataxin homologue Sen1 is required for efficient termination of RNA polymerase III transcription

“…It is tempting to speculate that such a release and catch-up mechanism could underlie the roles of fission yeast Sen1 in RNAP3 transcription: through translocation, Sen1 could nudge forward RNAP3 molecules that are weakly paused at gene-internal pause sites such as TFIIIC-binding sites and therefore facilitate transcription elongation, or release RNAP3 molecules that are stalled at primary terminator sequences to promote transcription termination. Importantly, the release and catch-up mechanism was also proposed to underlie the role of Mfd in both transcriptioncoupled repair and transcription-replication conflict resolution (Le et al, 2018) and budding yeast Sen1 has also been implicated in both transcription-coupled repair and transcriptionreplication conflict resolution (Mischo et al, 2011;Alzu et al, 2012;Brambati et al, 2018), strengthening the analogy with Mfd. However, the molecular mechanisms involved in such a release and catch-up mechanism might differ between Sen1 and Mfd, because Mfd was shown to translocate autonomously on double-stranded DNA, whereas both budding yeast and fission yeast Sen1 were shown to translocate on both single-stranded DNA and RNA, albeit at greater rate on DNA (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017).…”

Sen1 is required for RNA Polymerase III transcription termination in an R-loop independent manner

“…Importantly, the release and catch-up mechanism was also proposed to underlie the role of Mfd in both transcription-coupled repair and transcriptionreplication conflict resolution (Le et al, 2018). Strikingly, budding yeast Senataxin Sen1 has also been implicated in both transcription-coupled repair (Li et al, 2016) and transcription-replication conflict resolution (Alzu et al, 2012;Brambati et al, 2018;Mischo et al, 2011), strengthening the analogy with Mfd. A unifying way of interpreting the different roles of Senataxin Sen1 could therefore be to propose that it is targeted to chromatin through its interaction with RNA polymerases and that it subsequently patrols chromatin locally to facilitate the resolution of R-loops and/or stalled elongation complexes through a release and catch-up mechanism.…”