Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life1. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules1; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses2. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells3,4, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements5. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA–DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.
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We thank D. Garfinkel for plasmids pSM50, BDG606, BDG283, BDG102 and BDG598; K. D. Koh for strain KK-72; S. Y. Goo for construction of the YEp195SpGAL-RNH201 and YEp195SpGAL-rnh201(D39A) plasmids; S. Kowalczykowski for providing yeast Rad52 and RPA proteins; M. Fasken and A. Corbett for advice on the work and manuscript; B. Weiss, S. Balachander and C. Meers for critical reading of the manuscript; and all members of the Storici laboratory for assistance and feedback on this research. We acknowledge funding from the National Science Foundation grant number MCB-1021763 (to F.S.), the Georgia Research Alliance grant number R9028 (to F.S.) and the National Cancer Institute of the National Institutes of Health grant numbers CA100839 and P30CA056036 (to A.V.M.), for supporting this work. H.K. was supported by a fellowship from the Ministry of Science of Turkey.
The authors declare no competing financial interests.
Extended data figures and tables
a, Trans system on chromosome (Chr) III. HIS3 ATG and STOP codons are boxed. The HIS3 gene is disrupted by an insert (orange) carrying the artificial intron (AI). The consensus sequences of the AI are boxed. b, Trans system on chromosome XV. HIS3 ATG and STOP codons are boxed. The HIS3 gene is disrupted by an insert (yellow) containing the 124-base-pair homothallic switching endonuclease site (marked by lines). c, Cis system on chromosome III. HIS3 ATG and STOP codons are shown. The HIS3 gene is disrupted by an insert (orange) carrying the AI, which contains the 124 base pairs of the homothallic switching endonuclease site (yellow and marked by lines). The consensus sequences of the AI are boxed. *23-base-pair deletion of the AI, including the 5′ splice site, made in some strains.
Extended Data Figure 2 Efficient transcript-RNA-directed gene modification is inhibited by RNH201, requires transcription of the template RNA and formation of a DSB in the target gene.
a, Complementation of rnh201 defect suppresses transcript-RNA-templated DSB repair in cis-system rnh1 rnh201 spt3 cells. Wild-type (WT), spt3, rnh1 rnh201, rnh1 rnh201 spt3 strains of the cis system were transformed by a control empty vector (YEp195spGAL-EMPTY), a vector expressing catalytically inactive from of RNase H2 (YEp195spGAL-rnh201-D39A) or a wild-type form of RNase H2 (YEp195spGAL-RNH201). All the vectors have the galactose-inducible promoter. Shown is an example of replica-plating results (n = 6) from galactose medium to histidine dropout for the indicated strains and plasmids. b, Example of replica-plating results (n = 6) from galactose medium to histidine dropout for the indicated strains of the cis system, which have functional pGAL1 promoter and homothallic switching endonuclease (HO) gene, or have deleted pGAL1 promoter (pGAL1Δ), or deleted HO gene (hoΔ). c, Table with percentages of cells in the G1, S or G2 stage of the cell cycle out of 200 random cells counted for the indicated strains of the cis system after 0 h and 8 h from galactose induction. If a homothallic switching endonuclease DSB is made in his3, yeast cells arrest in G2, thus a high percentage of G2-arrested cells indicates occurrence of the homothallic switching endonuclease DSB. We also note that strains with spt3 mutation have a higher percentage of G2 cells than strains with wild-type SPT3 before DSB induction (0 h GAL). d, Results of qPCR of his3 RNA. Cells were grown in YPLac liquid medium O/N, and were collected and prepared for qPCR at 0, 0.25 or 8 h after adding galactose to the medium. Trans, blue bars; cis, red bars. Data are represented as a fold change value with respect to mRNA expression at time zero, as median and range of 6–8 repeats. The significance of comparisons between fold changes obtained at 0.25 h versus those obtained at 8 h, fold changes of different strains of the trans and cis systems, and between fold changes obtained in the trans versus cis system for the same strains at the same time point was calculated using the Mann–Whitney U-test and P values are presented in Supplementary Table 1jI, II and III, respectively. We note that an apparent higher level of his3 RNA is detected at 8 h in galactose in both trans- and cis-system rnh1 rnh201 cells relative to the other tested genetic backgrounds. Our interpretation of these results is that his3 RNA could be more stable in rnh1 rnh201 cells if present in the form of RNA–DNA heteroduplexes, and this may explain the increased frequency of His+ colonies observed in both trans and cis in the rnh1 rnh201 cells (Fig. 1c and Table 1a).
Extended Data Figure 3 Verification of his3 repair in trans- and cis-system rnh1 rnh201 spt3 cells via a homologous recombination mechanism using colony PCR.
a, Scheme of the trans system before DSB induction (BDI, groups of lanes 1 and 7) and after DSB repair (ADR, groups of lanes 2–6 and 8–12) with the primers used in colony PCR shown as small black arrows and named with roman numerals: I, HIS3.5; II, HIS3.2; III, INTRON.F; IV, HO.F. The primer pairs used for colony PCR are named A (I + II), B (I + III) and C (I + IV), and base-pair sizes of the expected PCR products are shown in brackets. b, Photos of agarose gels with results of colony PCR reactions. M, 2-log DNA ladder marker; the 100-, 300- and 500-base-pair band sizes are indicated by arrows. Groups of lanes 1 and 7, two isolates of trans-system rnh1 rnh201 spt3 mutants before DSB induction, each tested with primer pairs A, B and C. Groups of lanes 2–6 and 8–12, ten isolates of trans-system rnh1 rnh201 spt3 mutants after DSB repair, each tested with primer pairs A, B and C. c, Scheme of the cis system before DSB induction (BDI, groups of lanes 1 and 7) and after DSB repair (ADR, groups of lanes 2–6 and 8–12) with the primers used in colony PCR shown as small black arrows and named with roman numerals: I, HIS3.5; II, HIS3.2; III, INTRON.F; IV, HO.F. The primer pairs used for colony PCR are named A (I + II), B (I + III) and C (I + IV), and base-pair sizes of the expected PCR products are shown in brackets. d, Photos of agarose gels with results of colony PCR reactions. M, 2-log DNA ladder marker; the 100-, 300- and 500-base-pair band sizes are indicated by arrows. Groups of lanes 1 and 7, two isolates of cis system rnh1 rnh201 spt3 mutants before DSB induction, each tested with primer pairs A, B and C. Groups of lanes 2–6 and 8–12, ten isolates of cis-system rnh1 rnh201 spt3 mutants after DSB repair, each tested with primer pairs A, B and C.
Extended Data Figure 4 RNA-templated DNA repair occurs via homologous recombination and requires Rad52.
a, Scheme of the trans and cis his3/HIS3 loci in His− (before DSB induction) and His+ (after DSB repair) cells. The size of the BamHI (trans) or NarI (cis) restriction digestion products and the position of the HIS3 probe are shown. b, Photo of a ruler next to ethidium-bromide-stained agarose gel with marker and genomic DNA samples visible before Southern blot analysis. Lanes 1 and 14, 1-kilobase (kb) DNA ladder; 500-base-pair, 1-kb, 1.5-kb, 2-kb, 3-kb and 4-kb bands are indicated by arrows. Trans wild-type His− (lane 2) or His+ (lane 3), rnh1 rnh201 spt3 His− (lane 4) or His+ (lanes 5–7) cells, digested with BamHI restriction enzyme. Cis wild-type His− (lane 8) or His+ (lane 9), rnh1 rnh201 spt3 His− (lane 10) or His+ (lanes 11–13) cells, digested with NarI restriction enzyme. c, Southern blot analysis (same as in Fig. 2a, but displaying the entire picture of the exposed membrane) of yeast genomic DNA derived from trans wild-type His− (lane 2) or His+ (lane 3), rnh1 rnh201 spt3 His− (lane 4) or His+ (lanes 5–7) cells, digested with BamHI restriction enzyme and hybridized with the HIS3 probe, or derived from cis wild-type His− (lane 8) or His+ (lane 9), rnh1 rnh201 spt3 His− (lane 10) or His+ (lanes 11–13) cells, digested with NarI restriction enzyme and hybridized with the HIS3 probe. Lanes 1 and 14, 1-kb DNA ladder visible in the ethidium-bromide-stained gel (b). Sizes of digested DNA bands are indicated. The annealing reactions were promoted by either yeast Rad52 (d, e) or human RAD52 (f, g) (1.35 nM) in the presence or absence of RPA (2 nM) (yeast or human RPA was used in the reaction with yeast or human Rad52, respectively). In control protein-free reactions, protein dilution buffers were added instead of the respective proteins. dsDNA containing a protruding ssDNA tail (no. 508 and no. 509) was incubated with RPA (when indicated) and then Rad52 was added to the mixture. To initiate the annealing reactions, 0.3 nM 32P-labelled ssDNA (no. 211) or ssRNA (no. 501) were added. The reactions were carried out for the indicated periods of time, and the products of annealing reactions were deproteinized and analysed by electrophoresis in 10% polyacrylamide gels in 1× TBE at 150 V for 1 h. Visualization and quantification was accomplished using a Storm 840 Phosphorimager and ImageQuant 5.2 software (GE Healthcare). e, Treatment of RNA and DNA oligonucleotides with RNase. ssDNA (no. 211) or RNA (no. 501) (3 μM) was incubated with 100 μg ml−1 (or 7 U ml−1) RNase (Qiagen) in buffer containing 50 mM Hepes, pH 7.5 for 30 min at 37 °C, then 7% glycerol and 0.1% bromophenol blue were added to the samples and incubation continued for another 15 min at 37 °C before the samples were analysed by electrophoresis in a 10% (17:1 acrylamide:bisacrylamide) polyacrylamide gel at 150 V for 1 h in 1× TBE buffer. The gel was quantified using a Storm 840 Phosphorimager. The RNA oligonucleotide, but not the DNA oligonucleotide, is completely degraded by RNase.
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Keskin, H., Shen, Y., Huang, F. et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014). https://doi.org/10.1038/nature13682
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