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. 2015 May 5;112(18):5779-84.
doi: 10.1073/pnas.1501769112. Epub 2015 Apr 20.

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Ruth Stuckey  1 , Néstor García-Rodríguez  1 , Andrés Aguilera  1 , Ralf Erik Wellinger  2
Affiliations

Role for RNA:DNA hybrids in origin-independent replication priming in a eukaryotic system

Ruth Stuckey et al. Proc Natl Acad Sci U S A. .

Abstract

DNA replication initiates at defined replication origins along eukaryotic chromosomes, ensuring complete genome duplication within a single S-phase. A key feature of replication origins is their ability to control the onset of DNA synthesis mediated by DNA polymerase-α and its intrinsic RNA primase activity. Here, we describe a novel origin-independent replication process that is mediated by transcription. RNA polymerase I transcription constraints lead to persistent RNA:DNA hybrids (R-loops) that prime replication in the ribosomal DNA locus. Our results suggest that eukaryotic genomes have developed tools to prevent R-loop-mediated replication events that potentially contribute to copy number variation, particularly relevant to carcinogenesis.

Keywords: RNA:DNA hybrids; RNase H; replication; ribosomal DNA; topoisomerase 1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Lack of RNase H activities predominantly affects nucleolar DNA stability. (A) Tenfold serial dilutions of cells grown for 3 d on YPAD (control) or YPAD-containing 10 μg/mL CPT. (B) Rad52-YFP foci represent DNA repair centers associated to nuclear versus nucleolar DNA (rDNA; hatched). Nop1-mRFP was used as nucleolar marker. (Scale bars, 2.5 µm.) Percentage of cells containing Rad52-YFP foci counted in exponentially growing cells growing in the absence (control) or presence of CPT (10 µg/mL, 3-h treatment; Right). Data represent mean ± SD from three independent experiments. Note that the ratio of Rad52-YFP/Nop1-RFP colocalization increased from 29% (control) and 34% (+CPT) in WT cells to 61% (control) and 65% (+CPT) in r1∆ r2∆ mutants.
Fig. 2.
Fig. 2.
Two-dimensional gel analysis detects exceptional replication events in the rDNA of CPT-treated r1∆ r2∆ mutants. (A) FACS analysis of S-phase progression upon α-factor release of cells (WT, r1∆ r2∆) in the presence of 10 μg/mL CPT. G1 (n) and G2/M phase (2n) are indicated. (B) Schematic representation of major 2D gel signals including Y-, X-, and bubble-shaped molecules are displayed. The accumulation of replication intermediates at the RFB site as well as nonreplicating DNA (1×) are indicated. (C) Two-dimensional gel analysis of the BglII (B) digested rDNA locus. Probe A (Upper) detects the intergenic spacer region (IGS) containing the ribosomal origin of replication (ARS); the 5S gene, the RFB, and the 5′ end of the 35S gene. Replication pausing sites are indicated (open arrowheads). Probe B hybridizes to the RNAPI transcribed 35S gene. Replication intermediates indicative of bubble-shaped molecules are indicated (black arrowhead). The images are reduced by 5×. Quantification is shown in Fig. S2B.
Fig. 3.
Fig. 3.
Characterization of unscheduled replication intermediates in the rDNA. (Left) Comparison of r1∆ r2∆ and rrm3∆-dependent RFPs (indicated by lowercase letters a to d) detected by probe A. The open arrow marks a r1∆ r2∆ specific pausing site. (Right) A low-resolution mapping of the pausing sites based on their Y-arc location is shown. The images are reduced by 3.5×.
Fig. 4.
Fig. 4.
Top1AID*-depletion stimulates R-loop formation and unscheduled replication events in r1∆ r2∆ mutants. (A, Left) Western blot analysis of IAA (1 mM) induced Top1AID* protein degradation. Tir1 serves as loading control. Note that both proteins contain multiple Myc epitopes for detection. (Right) Quantification of RNA:DNA hybrids by S9.6 antibody detection in WT, r1∆ r2∆, and r1∆ r2∆ TOP1AID* mutants in the absence or presence of CPT or IAA, respectively (Right). (B) Two-dimensional gel analysis of RIs isolated from the r1∆ r2∆ TOP1AID* mutant cells grown in the presence of 1 mM IAA following α-factor release. The disappearance of the RFB signal is indicated (white asterisk). The images are reduced by 5.5×. Description as in Fig. 2.
Fig. 5.
Fig. 5.
RNAPI transcription is a prerequisite for the induction of unscheduled replication events. (A) Tenfold serial dilutions of indicated strains grown on YPAD (control) with or without 5 μg/mL CPT for 3 d at different temperatures allowing the permissive (23 °C) and semipermissive (30 °C) growth of rpa190-3 mutants (Rpa190 is the largest subunit of RNAPI). (B) Quantification of nuclear and nucleolar-associated Rad52-YFP foci in rpa190-3 r1∆ r2∆ mutants grown at indicated conditions. Data represent the mean ± SD from three independent experiments (**P < 0.001). (C) Two-dimensional gel analysis rpa190-3 r1Δ r2Δ triple mutant RIs isolated at 105 min after α-factor release in the presence of CPT at different temperatures. The presence of bubble-shaped molecules RI-derived from cells grown at 23 °C is indicated (black arrowhead). Description as in Fig. 2. (D) Model for TIR in yeast rDNA. RNA (red) and newly synthesized DNA (green) are indicated. The images are reduced by 5×. See text for explanation.
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