Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5′- to 3′-Helicase Activity with a Preference for Translocation on DNA Rather than RNA

Abstract

Background: Yeast Sen1 helicase and its human ortholog senataxin promote accurate transcription termination. Results: Sen1 helicase domain exhibits 5Ј-to 3Ј-helicase activity on DNA and RNA and binds endogenous RNA. Conclusion: Biochemical activities of purified Sen1 helicase domain are consistent with its proposed function in resolving cotranscriptional R-loops. Significance: Mutations in senataxin and its paralog IGHMBP2 cause crippling neurodegenerative diseases.

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

“…For structural studies, we expressed and purified a fragment of Sen1 encompassing residues 1,095–1,904 (hereafter referred to as Sen1 Hel ), with the shorter N‐terminus previously identified by the Brow's group (Martin‐Tumasz & Brow, 2015). Sen1 Hel showed levels of RNA‐dependent ATPase activity similar to those of full‐length Sen1 purified from yeast (ySen1 FL).…”

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

“…It was recently proposed that the Mfd translocase in Escherichia coli could both nudge forward weakly paused RNAP and induce the dissociation of stalled RNAP using a release and catch-up mechanism (Le et al, 2018). 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). 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 mechanisms involved probably differ in both species as budding yeast Sen1 contributes to RNAP2 transcription termination as part of the Nrd1-Nab3-Sen1 (NNS) complex, which is not conserved in human cells. Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017). Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017).…”

“…In addition, both budding yeast Sen1 and human Senataxin have been implicated in the repair of DNA damage Andrews et al, 2018;Cohen et al, 2018) and in the resolution of transcription-replication conflicts (Alzu et al, 2012;Richard et al, 2013;Yü ce & West, 2013). Both budding and fission yeast Sen1 can translocate in a 5 0 -3 0 direction on either single-stranded DNA or RNA in vitro (Kim et al, 1999;Martin-Tumasz & Brow, 2015;Han et al, 2017), and it is believed that long, co-transcriptional RNA-DNA hybrids (also known as R-loops) represent a critical substrate of budding yeast Sen1 and human Senataxin in vivo (Mischo et al, 2011;Skourti-Stathaki et al, 2011 andreviewed in Groh et al, 2017). Current models propose that the stabilization of R-loops upon Senataxin inactivation underlies the associated transcription termination and DNA repair defects.…”

Transcription Termination: Variations on Common Themes

“…In both cases the reaction can be fully recapitulated in vitro with purified components, demonstrating that a simple system containing only the helicase and the elongation complex is sufficient for transcription termination. Sen1 (like Rho) is an active helicase [83,84] and has been implicated in unwinding DNA:RNA hybrids (R loops) [85], but whether termination requires translocation on the nascent RNA and/or unwinding of nucleic acids duplexes is unclear. Deciphering whether Sen-1 dependent termination involves hybrid shearing, hyper-translocation or an allosteric change, as proposed for bacterial termination, remains a challenge for future studies.…”