Transcript-RNA-templated DNA recombination and repair

Abstract

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 life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. 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.

Extended Data Figure 1. DNA sequence of the his3 loci in the trans and cis systems

a, Trans system on 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 Chr XV. HIS3 ATG and STOP codons are shown. The HIS3 gene is disrupted by an insert (yellow) containing the 124-bp HO site (marked by lines). c, Cis system on Chr III. HIS3 ATG and STOP codons are shown. The HIS3 gene is disrupted by an insert (orange) carrying the AI, which contains the 124-bp of the HO site (yellow and marked by lines). The consensus sequences of the AI are boxed. * indicates a 23-bp 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 rnh1 rnh201 spt3 cells. 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 (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 and HO gene, or have deleted pGAL1, or deleted HO gene. c, Table with percentages of cells in the G1, S or G2 stage of the cell cycle out of random 200 cells counted for the indicated strains of the cis system after 0 h and 8 h from galactose induction. If an HO DSB is made in his3, yeast cells arrest in G2, thus, high percentage of G2 arrested cells indicates occurrence of the HO DSB. We also note that strains with spt3 mutation have higher percentage of G2 cells than strains with wild-type SPT3 before DSB induction (0 h GAL). d, Results of qRT-PCR of his3 RNA. Cells were grown in YPLac liquid medium O/N, and were collected and prepared for qRT-PCR at 0, 0.25 or 8 h after adding galactose to the medium. Trans strains have blue bars, cis red, respectively. Data are represented as a fold change value with respect to mRNA expression at time zero, as median with range of 6-8 repeats. The significance of comparisons between fold changes obtained at 0.25 h vs. those obtained at 8 h, fold changes of different strains of the trans and cis system, and between fold changes obtained in the trans vs. 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 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 rnh1 rnh201 spt3 cells via an HR 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 parentheses. b, Photos of agarose gels with results of colony PCR reactions. M, 2-Log DNA Ladder marker; the 100, 300 and 500-bp band sizes are pointed by arrows. Groups of lanes 1 and 7, two isolates of trans rnh1 rnh201 spt3 mutants BDI, each tested with primer pairs A, B and C. Groups of lanes 2-6 and 8-12, ten isolates of trans rnh1 rnh201 spt3 mutants ADR, 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 parentheses. d, Photos of agarose gels with results of colony PCR reactions. M, 2-Log DNA Ladder marker; the 100, 300 and 500-bp band sizes are pointed by arrows. Groups of lanes 1 and 7, two isolates of cis rnh1 rnh201 spt3 mutants BDI, each tested with primer pairs A, B and C. Groups of lanes 2-6 and 8-12, ten isolates of cis rnh1 rnh201 spt3 mutants ADR, each tested with primer pairs A, B and C.

Extended Data Figure 4. RNA-templated DNA repair occurs via HR 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 ruler next to ethidium bromide-stained agarose gel with marker and genomic DNA samples visible before Southern blot. Lanes 1 and 14, 1 kb DNA Ladder; 500 bp, 1 kb, 1.5 kb, 2 kb, 3 kb and 4 kb bands are pointed by arrows. Trans WT 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 WT 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 WT 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 WT 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 (panel b). Size of digested DNA bands is indicated by red arrows. The annealing reactions were promoted by either yeast (d,e) Rad52 or human (f,g) RAD52 (1.35 nM) in the presence or absence of yeast or human RPA, respectively (2 nM). In control protein-free reactions, protein dilution buffers were added instead of the respective proteins. To initiate the annealing reactions, 0.3 nM (molecules) ³²P-labeled ssDNA (#211) or ssRNA (#501) were added. The reactions were carried out for the indicated periods of time, and the products of annealing reactions were deproteinized and analyzed by electrophoresis in 10% polyacrylamide gels in 1 X TBE at 150 V for 1 h. Visualization and quantification was accomplished using a Storm 840 Phosphorimager. e,Treatment of RNA and DNA oligos with RNase. 3 μM ssDNA (#211) or RNA (#501) was incubated with 100 μg/ml (or 7 U/ml) 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 they were analyzed by electrophoresis in a 10% (17:1 acrylamide:bisacrylamide) polyacrylamide gel at 150 V for 1 h in 1 X TBE buffer. The gel was quantified using a Storm 840 Phosphorimager. The RNA oligo is completely degraded by RNase but not the DNA oligo.

Figure 1. Repair of a chromosomal DSB by transcript RNA

Scheme of the (a) trans and (b) cis cell systems used to detect DSB repair by transcript RNA. HO, HO endonuclease; AI, artificial intron; RT, reverse transcriptase. Examples of replica-plating results (n = 6) from galactose medium to histidine dropout medium demonstrating the ability of various yeast strains (relevant genotypes shown) of the trans and cis systems to generate histidine prototrophic colonies (c) in the absence of SPT3, or DBR1 function, or with PFA, (d) in the presence of the plasmid carrying the pGAL1-mhis3AI cassette (BDG606) or the control (BDG283), or (e) when the AI has a 23-bp deletion.

Figure 2. Transcript-templated DSB repair follows an HR mechanism

a, Southern blot analysis of yeast genomic DNA derived from trans WT 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 WT 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 (Extended Data Fig. 4a,c). Lanes 1 and 14, 1 kb DNA ladder visible in the ethidium bromide-stained gel (Extended Data Fig. 4b). Size of digested DNA bands is indicated by red arrows. b, Experimental scheme of Rad52-promoted annealing between RNA and DNA in vitro. Asterisk denotes ³²P label. ssDNA (#211) or ssRNA (#501) are in black, oligos #508 and #509 forming dsDNA are in blue and green, respectively. c, The kinetics of annealing promoted by yeast Rad52 and (d) human RAD52. Nucleoprotein complexes were assembled between either yeast or human Rad52 (1.35 nM) and tailed dsDNA (#508/509) (0.4 nM, molecules) in the presence (dashed lines) or absence (solid lines) of RPA (2 nM). Annealing was initiated by addition of ³²P-labeled ssRNA or ssDNA (0.3 nM, molecules). The kinetics of protein-free annealing reactions are indicated by open squares and circles. The error bars represent the standard error of the mean, n = 4. For the significance of comparisons between the last two time points we used the two-tailed Mann-Whitney U-test. P values are in Supplementary Table 1c.

Figure 3. Models of transcript RNA-templated DSB repair in cis

An actively transcribed DNA region experiencing a DSB uses its own transcript RNA as a bridging (a) or an extension (b) template for repair. The small black lines indicate initial annealing between the transcript RNA and the DSB end/s, and between the two DSB ends.