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. 2009 Oct;29(19):5203-13.
doi: 10.1128/MCB.00402-09. Epub 2009 Aug 3.

The S-phase checkpoint is required to respond to R-loops accumulated in THO mutants

Belén Gómez-González  1 , Irene Felipe-Abrio, Andrés Aguilera
Affiliations

The S-phase checkpoint is required to respond to R-loops accumulated in THO mutants

Belén Gómez-González et al. Mol Cell Biol. 2009 Oct.

Abstract

Cotranscriptional R-loops are formed in yeast mutants of the THO complex, which functions at the interface between transcription and mRNA export. Despite the relevance of R-loops in transcription-associated recombination, the mechanisms by which they trigger recombination are still elusive. In order to understand how R-loops compromise genome stability, we have analyzed the genetic interaction of THO with 26 genes involved in replication, S-phase checkpoint, DNA repair, and chromatin remodeling. We found a synthetic growth defect in double null mutants of THO and S-phase checkpoint factors, such as the replication factor C- and PCNA-like complexes. Under replicative stress, R-loop-forming THO null mutants require functional S-phase checkpoint functions but not double-strand-break repair functions for survival. Furthermore, R-loop-forming hpr1Delta mutants display replication fork progression impairment at actively transcribed chromosomal regions and trigger Rad53 phosphorylation. We conclude that R-loop-mediated DNA damage activates the S-phase checkpoint, which is required for the cell survival of THO mutants under replicative stress. In light of these results, we propose a model in which R-loop-mediated recombination is explained by template switching.

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Figures

FIG. 1.
FIG. 1.
Genetic interactions of THO mutations. (A) Viability of single and double mutants of THO and genes involved in checkpoint, replication, DNA repair, and chromatin remodeling. (B) Viability of single and double mutants of hpr1-101 and RFC-like genes involved in checkpoint. Single and double mutants were obtained by genetic crosses and tested for germination and growth in YPAD (except for the mrc1AQ strain, which was tested in minimal medium) at the restrictive temperature in the case of temperature-sensitive mutants and sensitivity to 150 mM HU and 0.02% MMS (or 5 mM HU and 0.005% MMS where there is an asterisk) by serial dilutions. Double mutants that grew worse than the corresponding single mutants in any of the conditions tested are highlighted in gray. WT, wild type; +++, normal growth; ++, mild growth defect; +, severe growth defect; −, no growth; nd, not determined. Unless a point mutation is specified with a number, all mutations used correspond to deletions with the kanMX4 cassette.
FIG. 2.
FIG. 2.
Genetic interaction of THO with checkpoint factors. (A) Synthetic growth defect of mRNP biogenesis and checkpoint factors. Seven representative tetrads of each genetic cross in which a synthetic growth defect was observed are shown. Double mutants are indicated by a white square. In the case of mec1Δ and rad53Δ, the sml1Δ mutation was necessary to avoid the lethality of the single mutants. (B) HU and MMS sensitivity of double mutants in hpr1Δ and RFC-like or PCNA-like factors in comparison to double mutants of hpr1Δ and mutations in the canonical replication factors RFC and PCNA. (C) hpr1-101 (hpr1-L586P) does not cause sensitivity to replicative stress even in combination with RFC-like factor mutants. Single- and double-mutant sensitivities to HU were determined by drop assays on rich medium without drugs (YPAD) or medium containing 150 mM HU unless otherwise indicated (*, 5 mM HU; #, 50 mM HU). WT, wild type.
FIG. 3.
FIG. 3.
High genetic instability in double mutants of HPR1 and RAD24. (A) Rad52 focus formation in asynchronously growing wild-type (WT), hpr1Δ, rad24Δ, and hpr1Δ rad24Δ cells. The median values and standard deviations of the results from three or four experiments performed with independent transformants are shown. (B) Recombination frequencies in wild-type, hpr1Δ, rad24Δ, and hpr1Δ rad24Δ strains measured with the LYΔNS system. The data represent the median values and standard deviations of the results from three or four fluctuation tests, each one performed with six independent colonies. (C) GCR rates in wild-type, hpr1Δ, rad24Δ, and hpr1Δ rad24Δ strains. The median values of the results from 6 to 12 independent experiments are shown. Diagrams of the different systems are shown at the top.
FIG. 4.
FIG. 4.
HU causes a mitotic cell cycle arrest in double mutants of THO and checkpoint factors. (A) Cell cycle progression in wild-type (WT), hpr1Δ, rad24Δ, and hpr1Δ rad24Δ strains (bar1Δ in all cases) in the absence (top panel) or presence of HU (bottom panel). Log represents asynchronous cell populations prior to the addition of α-factor. (B) HU sensitivity of the hpr1Δ rad24Δ mad2Δ, hpr1-101 (hpr1-L586P) rad24Δ mad2Δ, and hpr1Δ elg1Δ mad2Δ triple mutants in comparison to the respective simple and double mutants. (C) Rad52 focus formation in asynchronously growing mad2Δ, hpr1Δ mad2Δ, rad24Δ mad2Δ, and hpr1Δ rad24Δ mad2Δ cells. The median values and standard deviations of the results from three or four experiments performed with independent transformants are shown.
FIG. 5.
FIG. 5.
Rad53 phosphorylation in hpr1Δ cells. Western blot with JD148 antibody showing spontaneous Rad53 phosphorylation in wild-type (WT), hpr1Δ hpr1-101 (hpr1-L586P), hpr1Δ mrc1AQ, mrc1AQ, hpr1Δ rad9Δ, and rad9Δ strains.
FIG. 6.
FIG. 6.
Slowdown of replication fork progression through a chromosomal region in THO mutants. (A) Viability of wild-type (WT), hpr1Δ, and hpr1-101 (hpr1-L586P) mutants in the presence of high doses of replicative stress (200 mM HU). (B) A diagram of the PvuII, PstI, ClaI, and SpeI restriction sites on the chromosome V region surrounding ARS508 and the SPF1 gene. (C) 2-D gel migration pattern of replication intermediates of the 3.9-kb PvuII and 4.3-kb PstI DNA fragments from wild-type, hpr1Δ, and hpr1-101 (hpr1-L586P) cells (bar1Δ in all cases). An arrow indicates the accumulation of long Y molecules. A scheme of the expected replication intermediates and the corresponding migration pattern after 2-D gel electrophoresis is shown on top. (D) 2-D gel analysis of the replication intermediates of the 5.1-kb ClaI-SpeI DNA fragments from wild-type, hpr1Δ, and hpr1-101 (hpr1-L586P) (bar1Δ in all cases) cells in the presence of 200 mM HU at different times after release from α-factor. A scheme of the expected replication intermediates and the corresponding 2-D gel electrophoresis migration pattern is shown on top. (E) Effect of RNase H1 overexpression on hpr1Δ cell sensitivity to replicative stress. Wild-type and hpr1Δ cells were transformed with the multicopy plasmid pYGWRNH1, containing the RNH1 gene under the GAL1 regulable promoter. The growth of two independent transformants was tested in selective minimal medium with 0, 100, or 150 mM of HU and either glucose (SC-U) or galactose (SGal-U) as the carbon source.
FIG. 7.
FIG. 7.
Possible consequences of replication through cotranscriptional R-loops. RNA-DNA hybrids occurring on either the leading or the lagging strand might cause replication fork stalling with the concomitant accumulation of ssDNA, which is sensed by the S-phase checkpoints. These RNA-DNA hybrids may be efficiently removed or bypassed by a still unknown mechanism involving ribonucleases or putative DNA-RNA helicases (not shown). Nevertheless, when this stalling occurs within a DNA direct repeat, the RNA-DNA hybrid can be bypassed by a recombination-mediated replication process involving intermolecular template switching that implies the deletion of the intervening region. Template switching may also occur intramolecularly. A putative region containing a direct repeat (boxes) is shown.
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References

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