Abstract
Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization. We propose that Rrm3 and Pif1 promote genome stability at tDNAs by displacing the stable multi-protein transcription complex and by removing R-loops. Thus, we identify tDNAs as a new source of R-loop-mediated DNA damage. Given their large number and high transcription rate, tDNAs may be a potent source of genome instability.
Highlights
Saccharomyces cerevisiae encodes two Pif[1] family DNA helicases, Pif[1] and Rrm[3]
In S. cerevisiae, when replication and transcription move in opposite directions through a tDNA, forks slow even in wild type (WT) cells[12]
The abundance of Pif[1] and Rrm[3] was not affected by the absence of Rnh[1] (Fig. 1c). These findings suggest that the functions of both Pif[1] family helicases at tDNAs may be more important in cells with stabilized R-loops, as occurs in rnh1D cells
Summary
Saccharomyces cerevisiae encodes two Pif[1] family DNA helicases, Pif[1] and Rrm[3]. Rrm[3] promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). During each S-phase, the replication machinery encounters multiple classes of naturally occurring structures that impede fork progression These structures include stable protein complexes, highly transcribed genes and stable DNA secondary structures[1]. Stable protein–DNA complexes and R-loops are natural replication impediments[11] Both of these structures occur at tDNAs. In S. cerevisiae, when replication and transcription move in opposite directions through a tDNA, forks slow even in wild type (WT) cells[12]. A more than 3-fold increase in the size of the transcribed tDNA does not increase the size or extent of replication pausing at the gene These data suggest that Rrm[3] acts by promoting fork movement past the stable, multiprotein pre-initiation complex that is rapidly recycled such that many tDNAs are almost always pre-initiation complex associated 14,15. Rrm3D cells are not viable in the absence of at least two DNA helicases, Srs[2] and Sgs[1], but both of these helicases act after DNA replication, as the lethality in the double mutants (srs2D rrm3D and sgs1D rrm3D) is suppressed by deleting RAD51 (refs 25,26)
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