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. 2005 Mar 23;24(6):1267-76.
doi: 10.1038/sj.emboj.7600602. Epub 2005 Mar 3.

Impairment of replication fork progression mediates RNA polII transcription-associated recombination

Félix Prado  1 , Andrés Aguilera
Affiliations

Impairment of replication fork progression mediates RNA polII transcription-associated recombination

Félix Prado et al. EMBO J. .

Abstract

Homologous recombination safeguards genome integrity, but it can also cause genome instability of important consequences for cell proliferation and organism development. Transcription induces recombination, as shown in prokaryotes and eukaryotes for both spontaneous and developmentally regulated events such as those responsible for immunoglobulin class switching. Deciphering the molecular basis of transcription-associated recombination (TAR) is important in understanding genome instability. Using novel plasmid-borne recombination constructs in Saccharomyces cerevisiae, we show that RNA polymerase II (RNAPII) transcription induces recombination by impairing replication fork progression. RNAPII transcription concomitant to head-on oncoming replication causes a replication fork pause (RFP) that is linked to a significant increase in recombination. However, transcription that is codirectional with replication has little effect on replication fork progression and recombination. Transcription occurring in the absence of replication does not affect either recombination or replication fork progression. The Rrm3 helicase, which is required for replication fork progression through nucleoprotein complexes, facilitates replication through the transcription-dependent RFP site and reduces recombination. Therefore, our work provides evidence that one mechanism responsible for TAR is RNAP-mediated replication impairment.

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Figures

Figure 1
Figure 1
Homologous recombination is induced by RNAPII-mediated transcription if this occurs in the opposite direction to an oncoming replication fork. (A) Schemes of the centromeric plasmids harbouring the recombination constructs GAL-IN and GAL-OUT, and of the LEU2 recombination product. The arrows indicate the progression orientation of RNAPII transcription driven from the GAL1 promoter and of the replication forks initiated at ARSH4. The distance that each fork has to traverse from ARSH4 to the promoter are approximately 2.5 and 6 kb for the rightward- and leftward-advancing forks, respectively. (B) Northern analysis of transcripts emerging from the direct-repeat constructs in wild-type cells grown either in glucose (Glu; transcription OFF) or galactose (Gal; transcription ON). (C) Recombination frequencies in wild-type and rad52Δ strains. The average and standard deviation are indicated.
Figure 2
Figure 2
Recombination is induced by transcription only if this is active during S phase. (A) Schemes of the CLN-IN, HHO-IN, HHF-IN and HHF-OUT constructs. The leu2 direct repeats are under the control of the CLN2, HHO and HHF2 promoters, respectively, which are activated in G1 phase in the CLN-IN construct and in late G1/S phase in the HHF-IN and HHO-IN constructs. (B) Recombination frequencies of each construct in the wild-type strain. The average and standard deviation are indicated. (C) Northern analysis of leu2 transcripts emerging from each system (leu2s). RNA levels are normalized with respect to the CLN-IN values, taken as 1. (D) Run-on analysis of leu2 transcripts emerging from the CLN-Leu and HHF-Leu constructs. A scheme of these constructs, which are driven by the CLN2 and HHF2 promoters, respectively, is shown on top. The amount of mRNA bound to each probe is normalized with respect to the CLN-IN values, taken as 1. lacZ was used as negative control.
Figure 3
Figure 3
Converging transcription and replication leads to a 2D-gel-detectable RFP. (A) Schematic representation of the migration pattern of the Y-, bubble-, double Y- and X-shaped replication intermediates in 2D-gel electrophoresis. n indicates unreplicated molecules, RIs replication intermediates and nd not determined molecules. (B) 2D-gel analysis of the SalI–ScaI restriction fragment covering the leu2 repeats in the CLN-IN and HHF-IN constructs. Note that bubble molecules are not detected due to the terminal location of ARSH4 in the SalI–ScaI fragment. (C) 2D-gel analysis of the ScaI restriction fragment covering the leu2 repeats in the GAL-IN (either from cells grown in glucose (Glu) or galactose (Gal)), CLN-IN and HHF-IN constructs. The location of ARSH4 in an internal position leads to the detection of a bubble arc in addition to the Y-arc. A scheme of the replication intermediates expected for each restriction fragment and the position of the transcription-dependent RFP in the HHF-IN and GAL-IN constructs (solid arrow) is shown on the left.
Figure 4
Figure 4
Analysis of the progression of replication forks that are codirectional with transcription. (A) 2D-gel analysis of the ScaI restriction fragment covering the leu2 repeats in the GAL-OUT construct isolated from cells grown in glucose (Glu; transcription OFF) or galactose (Gal; transcription ON). (B) 2D-gel electrophoresis of the SalI–NdeI restriction fragment covering the leu2 repeats in the GAL-OUT and HHF-OUT constructs isolated from cells grown in galactose or glucose, respectively. An open arrow indicates a weak transcription-dependent RFP in the HHF-OUT construct. The asterisks indicate the expected position corresponding for the RFP at the leu2Δ3′ repeat as detected in the GAL-IN and HHF-IN constructs (Figure 3). Note that the signal accumulated in the inflection points of the Y-arcs in the GAL-OUT construct is the consequence of the overlapping left and right arcs, and does not correspond to a true RFP, since it does not change its position in the overlapping fragments.
Figure 5
Figure 5
The transcription-dependent RFP is independent of direct repeats. 2D-gel analysis of Leu+ recombinants from the HHF-IN construct containing just one repeat unit (HHF-Leu construct). The overlapping ScaI (left) and SalI–ScaI (right) restriction fragments are shown on top of each panel. A solid arrow indicates the RFP.
Figure 6
Figure 6
Increase of transcription-dependent RFP and recombination in the absence of the Rrm3 helicase. (A) 2D-gel analysis of the ScaI restriction fragment covering the leu2 repeats in the GAL-IN, HHF-IN and CLN-IN constructs isolated from rrm3Δ cells grown in glucose. Solid arrows indicate the transcription-dependent RFP in the HHF-IN construct. (B) 2D-gel analysis of the SalI–NdeI restriction fragment covering the leu2 repeats in the GAL-OUT and HHF-OUT constructs isolated from rrm3Δ cells grown in glucose. Open arrows indicate the transcription-dependent RFP in the HHF-OUT construct. (C) Recombination frequencies of the GAL-IN, CLN-IN, HHF-IN, GAL-OUT and HHF-OUT constructs in the wild-type and rrm3Δ strains grown in glucose. The average and standard deviation are indicated.
Figure 7
Figure 7
The transcription-dependent RFP in the HHF-IN construct is not affected by rad51Δ, rad52Δ, sgs1Δ and srs2Δ. 2D-gel analysis of the ScaI restriction fragment covering the leu2 repeats in the HHF-IN construct in wild type, rad51Δ, rad52Δ, sgs1Δ and srs2Δ. Solid arrows indicate the transcription-dependent RFP at the leu2Δ3′ repeat. Quantification data of the RFPs relative to the replication intermediates, taken as 100, are shown.
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