Yeast Sen1 Helicase Protects the Genome from Transcription-Associated Instability

1In S. cerevisiae nascent transcripts formed by RNA polymerase II (Pol II) on protein-coding genes are immediately processed, packaged, and exported to the cytoplasm. Messenger RNA (mRNA) packaging protects transcripts from degradation, but also the DNA template from invasion of nascent RNA into the DNA duplex behind elongating Pol II. The resulting RNA:DNA hybrid exposes single stranded (ss) nontemplate DNA, a structure referred to as an R loop. R loop formation has been associated with increased occurrence of transcription-associated mutation (TAM) or recombination (TAR), presumably because both induced and spontaneous lesions are more likely to occur on ssDNA. Thus, deletion of genes encoding the THO (Thp2, Hpr1, Mft1, and Tho2) and THSC or TREX-2 (Thp1, Sac3, Sus1, and Cdc31) complexes required for mRNP formation in S. cerevisiae—or, similarly, the splicing factor ASF/SF2 in metazoans—increase levels of R loop formation and consequently TAM and TAR. R loop formation in these mutants may also be connected to Pol II stalling, consequently interfering with processive elongation and RNA processing. Similarly, DNA replication may be compromised when replication forks encounter R loops or a stalled Pol II.

2Although little is known about R loop resolution in yeast, in mammals their formation and resolution play a productive role in the stimulation of class switch recombination (CSR) and somatic hypermutation (SHM) in clonally expanding B cells. Both processes are initiated by activation induced deaminase (AID). Double-strand breaks (DSBs) subsequently trigger CSR via nonhomologous end joining (NHEJ). Although S. cerevisiae does not express AID, ectopically expressed AID can recognize ssDNA in R loops as a substrate when expressed in mRNA packaging mutants.

3Many events during transcription are orchestrated by proteins binding to the carboxy-terminal domain (CTD) of the Pol II largest subunit. CTD consists in yeast of 26 hepta-peptide repeats (YSPTSPS) that are dynamically modified during transcription. In particular, serine phosphorylation occurs during early (ser5-, 7-P) and late (ser2-P) elongation phases to allow stage specific binding of elongation and RNA processing factors. Transcription termination is also directed by different CTD-bound proteins that recognize specific sequences on the emerging nascent RNA. For protein-coding genes, this requires polyA (pA) site recognition by a ser2-P CTD bound multicomponent cleavage and polyadenylation complex (CF IA/B and CPF), as well as degradation of the downstream RNA by Rat1 exonuclease.

4Termination of many noncoding RNAs requires an additional component, the NRD complex (Sen1, Nab3, and Nrd1), in which Nrd1 is bound to ser5-P CTD. NRD-dependent termination also requires recognition of frequent short RNA sequences by Nrd1 and Nab3 (GUAA/G and UCUU respectively), although the exact sequence and NRD component requirements may vary for different terminators. Nrd1 ser5-P CTD specificity confines this termination pathway to transcriptional stages in which ser5-P CTD prevails and leaves promoter distal Nrd1/Nab3 binding sites unrecognized. Furthermore, as Nrd1 interacts with the exosome, NRD-terminated RNA is either degraded to protein protected stable transcripts (e.g., snoRNAs) or completely, as is the case with cryptic unstable transcripts (CUTs). Recent genome-wide transcription profiling studies reveal the wide extent of CUTs produced by Pol II and terminated by NRD. This further emphasizes the biological importance of NRD-dependent termination. Importantly, both termination pathways can substitute for each other and so provide mutual fail-safe termination mechanisms. Thus, NRD termination is also important to rescue polymerases that fail to terminate at a polyA signal, especially on highly transcribed genes. Interestingly, these genes show a particular requirement for Sen1.

5SEN1 codes for a 240 kDa superfamily I helicase, and its S. pombe homolog possesses 3′-5′ nucleic acid unwinding activity. The essential C terminus contains the helicase domain, a nuclear localization sequence (NLS), and a domain necessary for interaction with the Glc7 phosphatase component of CPF. The Sen1 975 N-terminal amino acids, although dispensable for growth, interact with Pol II, RNase III endonuclease Rnt1, and the nucleotide excision repair endonuclease Rad2. Mutation of the Sen1 helicase domain results in direct and indirect pleiotropic defects in transcript processing and termination, leading to a perturbed genome-wide profile of Pol II and defective Pol I transcription termination. Although the severe character of these phenotypes may be explicable by the limiting presence of Sen1 in NRD (as it is only present at 125 copies/cell), they have not been clearly attributed to a molecular function of Sen1. Employing the temperature sensitive sen1-1 mutant (helicase domain G1747D), we set out to characterize the molecular role of Sen1 in transcription termination. We now identify broad functions for Sen1 during Pol II transcription in reducing R loop formation and consequent prevention of transcription-associated genome instability.

6Mutation of the Sen1 helicase domain results in genome-wide transcription termination defects of noncoding RNAs, but also of some protein coding genes. Thus, when tested by transcription run on (TRO) experiments with the plasmid gene construct KGG (Figure 1A), with the KanMX4 gene terminated by the weak GAL10 pA signal, sen1-1 mutants grown for 150 min at nonpermissive temperature (37°C) show a strong termination defect (Figure 1A, upper panels). This indicates either a requirement for Sen1 in Rat-dependent termination or that some transcripts over the weak GAL10 pA signal are terminated by the NRD failsafe termination mechanism.

7To determine whether Sen1 protein-protein interactions or its helicase function are required for transcription termination, we repeated TRO analysis in WT and sen1-1 cells transformed with additional Sen1 expression constructs. Transcribed from an ACT1 promoter, these either contained the NLS and the Glc7 interaction domain [Sen1(323)] or additionally the C-terminal helicase domain [Sen1(1212)]. As shown in Figure 1A, Sen1(1212) but not Sen1(323) rescued the sen1-1 termination defect, implying that the sen1-1 termination defect is caused by loss of helicase function and not Glc7 mediated recruitment of CPF. We also examined steady-state mRNA produced from the KGG construct (Figure 1B). mRNA levels were reduced in sen1-1 cells and partially complemented by coexpression of Sen1(1212), but not Sen1(323). Similarly, coexpression of Sen1(1212) restored wild-type levels of endogenous PMA1 mRNA, also previously shown to display mild termination defects in sen1-1.

8The above results indicate that the Sen1 helicase domain is required both for efficient Pol II termination and mRNA accumulation. As these effects could be attributed to defective 3′ end processing, we employed an in vitro cleavage and polyadenylation assay using CYC1 3′ flanking RNA as the pA substrate (Figure 1C). sen1-1 shows no defects in RNA 3′ end processing. Confirmation of this result is provided by reverse transcription analysis of ACT1 pA usage, in which sen1-1 showed WT pA selection (Figure S1 available online). In contrast, a CF IA mutant strain, rna14-1, showed the expected defect in both in vitro 3′ end processing and in vivo pA selection (Figure 1C and Figure S1). Finally, like sen1-1, the rat1-1 termination mutant (or both combined) had no effect on mRNA 3′ end formation but stabilized the 3′ end cleavage product, indicative of loss of exonuclease “torpedo” function. Overall, these combined analyses show that the Sen1 helicase is dispensable for 3′ transcript processing but is required to promote transcriptional termination.

9Since S. pombe Sen1 can use RNA:DNA hybrids as an in vitro substrate, we considered the possibility that Sen1 may remove RNA:DNA hybrids formed by nascent RNA and the template strand. Such hybrids were previously shown to form in THO mutants, causing increased rates of transcription associated mitotic recombination. RNA:DNA hybrids may also be naturally encountered in transcribed regions downstream of pA signals, where THO is undetectable on chromatin. We therefore tested whether sequences downstream of a pA signal elicit TAR in sen1-1. We employed a plasmid borne recombination substrate that carries two truncated regions of LEU2 overlapping by 600 nt of homologous sequence (LNA). Lack of THO elicits TAR in LNA and consequent restoration of LEU2, as previously shown (Figure 2A). In contrast, when transcription between both repeats is terminated by insertion of the CYC1 38nt pA signal (CYC1t, LNAT), recombination levels in the hpr1Δ strain were reduced to background WT levels, presumably because Pol II termination restricts R loop formation. Similar analysis of LNA and LNAT transformed rat1-1 and rna14-1 showed no detectible increase in recombination, confirming that defects in CPF/Rat1 dependent transcription termination per se do not promote recombination. In marked contrast, sen1-1 transformed with either LNA or LNAT showed high levels of recombination, suggesting that RNA:DNA hybrids may form throughout the mRNA coding region irrespective of the CYC1t. This lack of CYC1t suppression reiterates the sen1-1 CYC1 termination defect previously reported. Moreover, the fact that CYC1t (in LNAT) further stimulated recombination may reflect an increase in R loop formation downstream of pA signals.

10To determine whether this recombination phenotype was specific to Sen1, we similarly tested other NRD complex components. Although recombination levels were somewhat increased in nab3 and nrd1 CTD-interacting domain mutants (but not the RNA binding domain mutant nrd1-102) transformed with LNA, they were reduced to background levels in LNAT (Figure 2A). This suggests that these NRD mutants still recognize the CYC1t. Why these NRD mutations elicit some recombination is unclear at this point, but may reflect alteration in mRNP biogenesis. The fact that CYC1t abrogates recombination in NRD mutants but stimulates recombination in sen1-1 clearly separates Sen1 function from Nab3 and Nrd1 and argues that Sen1 plays a distinct role outside the NRD complex.

11Hyperrecombination in THO and THSC/TREX-2 mutants shows clear transcription dependence, as it increases with greater transcript length and transcription rate but decreases when the R loop-forming RNA is removed either by RNase H activity or ribozyme directed RNA cleavage. To test whether sen1-1 shows a similar transcription-dependent recombination phenotype, we analyzed sen1-1 recombination levels for various direct-repeat recombination substrates. As shown in Figures 2B and 2C, levels of recombination in sen1-1 correlate with the length and transcriptional rate of the gene. Thus, TAR, although not abolished, was significantly decreased in two different recombination substrates when transcription from a GAL1 promoter was glucose repressed. Finally, we verified that recombination was also stimulated between direct repeats in a chromosomal context (Figure 2D). These observations suggest that there is a correlation between transcriptional activity and sen1-1 recombination levels.

12Evidence for RNA:DNA hybrid accumulation in THO mutants derives from expression of the human AID in yeast which was shown to cause a 25-fold increase in TAR. Employing 5′ and 3′ truncated overlapping GFP repeats and intervening LacZ sequence as a recombination substrate, FACS analysis of GFP-positive cells showed that AID expressed in sen1-1 also stimulates recombination albeit only 2.5-fold (P[Wilcoxon-rank-sum test] = 0.017; Figure 3A). As discussed below, this moderate but significant stimulation of TAR by AID could reflect the nature of RNA:DNA hybrids formed in sen1-1 or be due to the fact that recombination levels in sen1-1 cells that did not express AID were already very high.

13AID C to U deamination preferentially occurs in a WRC (or GYW on the opposite strand) sequence. In regions of R loop formation, AID has access to the nontranscribed strand (NTS), although some mutations occur on the transcribed strand (TS). In an attempt to analyze the nature of AID-induced mutations in sen1-1, we transformed WT and sen1-1 cells (both ura3–) with plasmid-encoded LACZ::URA3 chimeric gene (pLAUR, Figure 3B) and selected AID-induced ura3 mutations on 5-FOA (5-fluoorotic acid). Although mutation rates were very low in WT and sen1-1 cells grown at semipermissive temperature, AID expression in sen1-1 significantly increased mutation rates (Figure 3B). DNA sequencing of 57 5-FOA-resistant transformants revealed that 42% carried a point mutation in URA3, of which 71% had a point mutation within an AID sequence motif. Of these mutations 70% occurred on C (12; Fisher's test; p < 0.003) or the NTS and only 30% (five; p < 0.05) occur on G or the TS (Figure 3C and Figure S3). The small amount of WT transformants sequenced showed a distribution as earlier reported. Since AID expression not only increased the amount of point mutations within the transcribed URA3 in sen1-1 but also preferentially acted on the NTS, this suggests that R loops are formed during transcription in sen1-1 cells and displace the nontranscribed DNA strand.

14We next tested whether the high recombination levels seen in sen1-1 were sensitive to RNase H overexpression as previously observed with THO and THSC/TREX2 mutants and indicative of increased R loop formation. Thus, RNase H overexpression (from pRNH201) reduced recombination rates of pLLacZ in sen1-1 by 5.6-fold (Figure 3D). Furthermore, the sen1-1 recombination phenotype correlated with deficient helicase activity and could be rescued by high copy expression of pYsen1, encoding for the Sen1 helicase domain. To exclude the possibility that increased recombination levels in sen1-1 could be a consequence of the sen1-1 transcription termination defect, rather than the ability of Sen1 to remove R loops, we tested the capability of Sen1 helicase domain to suppress the hyperrecombination phenotype of the THO mutant mft1Δ, which displays no transcription termination defect but increased levels of R loops. As shown in Figure 3E, overexpression of the Sen1 helicase domain in AID- and pGLG-transformed mft1Δ cells caused a substantial reduction in the number of GFP-recombinant cells. We conclude that Sen1 enzymatic activity is able to directly restrict cotranscriptionally formed R loops.

15To obtain independent evidence for the existence of R loops formed in sen1-1, we employed both DNA (DIP; Figure 4A) and chromatin immunoprecipitation analysis (ChIP; Figure S4A) with the RNA:DNA hybrid-specific antibody (S9.6). Yeast transformed with pGLLacZ displayed hybrid signal over the recombining LEU2 sequence in sen1-1 but not WT cells when shifted for 1 hr to nonpermissive temperature and induced by galactose. Hybrid signal then diminished to WT background levels when transcription was repressed by glucose addition to the medium (Figure 4A). Notably, the untranscribed origin region of pGLLacZ gave background signals (Figure S4B). Furthermore, where DIP signals were detected on LGZ2 and LGZ5 in sen1-1 grown in galactose, these signals were sensitive to RNase H digestion prior to immunoprecipitation (Figure S4C). Overall, these data demonstrate that R loop formation is highly dynamic, closely following transcriptional activation and repression of pGLLacZ. Parallel experiments with the THO mutant strain hpr1Δ gave significant but lower levels of hybrid signal over the 5′ positioned LEU sequence (LGZ2) but not over LGZ5 (Figure 4A). The 3′ LGZ5 probe spans the LEU2 pA signal and so will lack transcripts in hpr1Δ due to Pol II termination. sen1-1 in contrast is termination defective so that read-through transcripts in this strain will still elicit R loop formation.

16The S9.6 antibody was also employed in a regular ChIP analysis of endogenous PMA1 and compared to Pol II ChIP profiles. RNA:DNA hybrid signal was detected over PMA1 (Figure S4A), although general signal intensities were lower than those obtained by DIP analysis of the highly expressed pGLLacZ. When normalized to the gene 5′ end in WT and sen1-1 cells, signals were detected wherever Pol II was present on chromatin. Of note, in chromatin isolated from rna14-1 that shows no recombination phenotype, little hybrid could be detected downstream of the pA signals, even through Pol II is still present at these positions. This may indicate that while hybrid is degraded downstream of the pA signal in presence of Sen1 and Rat1, it is stabilized in rat1-1 sen1-1 and to a lesser extent in sen1-1. A modest accumulation of hybrid signal was observed over the pA signal compared to the gene body (P6 versus P5) in all tested strains and may indicate a region that is particularly prone to form RNA:DNA hybrids.

17In view of the only modest RNA:DNA hybrid accumulation observed over PMA1, we developed an independent assay for R loop formation over this same gene (Figure 4B). Genomic DNA with associated nascent transcripts was isolated from WT and sen1-1 cells, shifted for 1 hr to 37°C, and subjected to RNase H digestion. Subsequently, DNA was digested with DNase I and nascent RNA that survived this treatment was detected by RT-PCR with RT primers either within PMA1 (P5) or downstream of the pA signal (P7). As shown in gel fractionation of these amplified DNAs, sen1-1 chromatin associated RNA is highly sensitive to RNase H treatment. Quantitative PCR (qPCR) values (ratio of RNase H sensitive to total signal) show that although in sen1-1 chromatin associated RNA is nearly fully RNase H sensitive, in WT RNase H sensitivity increases downstream of the PMA1 pA signal. This suggests that in sen1-1 a higher fraction of RNA forms R loop structures with genomic DNA, whereas in the WT R loops are particularly prone to forming downstream of the pA site.

18From these combined analyses, we conclude that R loops accumulate in sen1-1 in a transcription-dependent manner. Furthermore, they imply that WT-transcribed and subsequently packaged RNA still forms some level of RNA:DNA hybrids. The amount of RNA in R loop conformation appears to differ throughout the transcribed PMA1 gene, with a particular prevalence over the pA site.

19Experiments presented so far suggest that R loops formed in sen1-1 elicit TAR. However, to substantiate this conclusion and further exclude the possibility that sen1-1 TAR is an indirect consequence of its transcription termination defect, we employed genomic analysis. A comparison of RNA steady-state levels of sen1-1 to WT cells grown for 150 min at 30°C was performed by hybridization to strand-specific tiling arrays (data available at http://www.ebi.ac.uk/arrayexpress/ with accession number E-TABM-863). This revealed that among the transcripts whose expression was significantly changed (adjusted p value of < 0.01), stable unannotated transcripts were overrepresented (19% stable unannotated transcripts versus 7% for open reading frames [ORFs]), confirming a Sen1 role in the termination of some of these genes (Figure S5A and Table S4). Importantly, THO or THSC genes were absent among the 347 ORFs that were significantly changed, excluding the possibility that R loops are formed in sen1-1 as an indirect consequence of alteration in THO or THSC/TREX-2 expression. We next considered whether the 347 significantly changed ORF transcript levels correspond to DNA damage repair and cell-cycle (DDCC) genes. DDCC genes were underrepresented, and among those changed, no clear trend was observed (Figure S5B and Table S4), with some mildly upregulated (i.e., REC104, POL4, SMC5, SCM4, and TOP2) and others downregulated (i.e., DIA2, MMS2, and NEJ1).

20To define which DNA repair mechanism was induced by R loops formed in sen1-1, we searched for synthetic genetic interactions of sen1-1 with mutants of candidate genes involved in either NHEJ or homologous recombination (HR). At permissive temperature (25°C) or in presence of replicative stress, we observed genetic interaction with various factors involved in HR but not in NHEJ (Figure 5). Thus, sen1-1 (but not nrd1-102; Figure S5C) causes synthetic lethality with rad50 and mre11 deletion mutants, both found in a complex with Xrs2 and involved in initial recognition and ss resection at a DSB (Figure 5A). The critical need for HR in sen1-1 cells is demonstrated by the phenotypes of double mutants of sen1-1 and rad52Δ, sgs1Δ, srs2Δ, or mus81Δ. These all showed synthetic defects or displayed increased sensitivity to growth in hydroxyurea (HU). In contrast, sen1-1 yku70Δ double mutants showed neither growth defects nor increased sensitivity to HU, suggesting that NHEJ is not required for cell survival of sen1-1 cells (Figure 5B). Taken together, these data suggest that proteins involved in HR but not NHEJ are important to maintain sen1-1 viability.

21In cells that accumulate DNA damage, factors involved in DNA repair are rapidly recruited to the damage site. Consistent with the role of R loops in DNA damage as seen in sen1-1, we observed that many sen1-1 cells display an accumulation of GFP-tagged Rad52 (encoded on pWJ144; Figure 6A). This percentage of sen1-1 cells forming foci increased from 8% at 25°C to 13% when cultures were shifted to semipermissive (30°C) or nonpermissive (37°C) temperature for 3 hr. In contrast, only about 1% of WT cells formed foci under these conditions. Importantly, steady-state RNA analysis from the same cell populations showed that Rad52 foci formation correlated with accumulation of a bicistronic transcript from the SNR13 snoRNA gene, which results from termination at the next available pA when NRD-dependent termination is defective. As shown in Figure 6A (right panels), the SNR13-TRS31 transcript is already present in sen1-1 at 25°C but increases over time at 30°C and to a greater extent at 37°C. These results connect the various sen1-1 phenotypes observed in this work and support the assumption that they are caused by mutation of Sen1 helicase domain in sen1-1.

22Using this assay as an indicator of ongoing HR in sen1-1, we reinvestigated the role of Sen1 helicase in preventing or resolving transcription dependent R loops. WT and sen1-1 transformed with Rad52-GFP and pYSen1, which encodes the Sen1 helicase domain transcribed from the GAL1 promoter, were grown in raffinose as a neutral carbon source for 1 hr at 30°C. After addition of glucose or galactose to respectively repress or activate Sen1 helicase expression, the amount of Rad52 foci forming cells was counted. Expression of the Sen1 helicase domain rapidly reduced the number of sen1-1 nuclei displaying Rad52 foci to almost WT levels (Figure 6B). Employing a sen1-1 rpb1-1 double mutant, which allows rapid transcription shutdown at the nonpermissive temperature due to a mutation in the Pol II largest subunit (rpb1-1) (Figure 6C), we could also correlate the formation of Rad52 foci to transcriptional activity. When grown at 37°C, Rad52-GFP transformed sen1-1 rpb1-1 cells displayed a time-dependent decrease in Rad52 foci as compared to growth at 25°C. Since sen1-1 cells accumulate Rad52 foci when shifted to 37°C, these data indicate that upon transcription shutdown, Rad52 foci disappear either as DNA damage is immediately repaired or the cells die.

23The deleterious effects of transcription on genome integrity have been suggested by various observations. Whenever the integrated process of transcript processing, packaging, and export in eukaryotes is disrupted, genome instability can be observed. This has been shown to derive from R loops, which preferentially form when mRNP biogenesis is disrupted. Our data show that Sen1 helicase plays a pivotal role in the prevention of genome instability by recombination. A large fraction of this instability is transcription dependent and linked to the formation of R loop structures. The exact nature of these structures remains to be established, but we show their accumulation can still occur with normal mRNP biogenesis. If these structures are not removed by either Sen1 helicase or RNase H directed degradation, they can exert a deleterious effect on genome stability, as is further illustrated by SEN1 genetic interaction with HR genes. The occurrence of Rad52 foci, as a marker for ongoing recombination, shows that recombination is related to transcription, as well as to the presence of a functional Sen1 helicase domain. In summary, we suggest that R loop formation is more frequent than hitherto anticipated and requires active removal by helicases such as Sen1.

24We suggest that as soon as the nascent transcript emerges from the polymerase body, mRNA packaging and R loop formation occur in kinetic competition (Figure 7). A fragile equilibrium between protective mRNA packaging and the hiding of specific recognition sequences is likely to exist. Therefore, RNA packaging is likely to be incomplete so leaving some transcript available for R loop formation. For pervasive CUT transcription, termination depends on NRD and by inference on Sen1. If R loops formed in sen1-1 extend to many CUT loci, then their accumulation, even if transient, would cover substantial regions of the genome. In both CUTs and mRNA coding genes, R loops could interfere with DNA replication, induce ssDNA breaks, or be recognized as recombination intermediates. Any of these possibilities could explain the essential need for DSB sensing proteins in sen1-1 (Figure 5). However, the different genetic interactions of sen1-1 and hpr1 with HR or S phase checkpoint genes suggest structural and functional differences of the replication/recombinogenic intermediates that are formed. Alternatively, these differences may hint at a transcription-independent role of Sen1 in DNA damage repair that is yet to be uncovered.

25How may R loop accumulation in sen1-1 be related to its transcription termination defect? R loops were originally hypothesized to slow down transcription elongation, thereby enhancing termination. This would give time for the Rat1 5′-3′ exonuclease “torpedo” to catch up with Pol II but would require R loop resolution by an enzymatic activity such as Sen1 prior to degradation. Based on observations made on THO mutants, R loops have been suggested to interfere with transcription elongation. Employing sen1-1, in which transcript processing is normal, we predict that reduced steady-state RNA accumulation is due to reduced transcript elongation. Furthermore, the data presented here support the view that R loops preferentially form in termination regions. Thus, we employed the LNA/LNAT recombination substrates, anticipating that even though the CYC1 pA would not elicit termination, it should serve as a 3′ processing signal (Figure 1C), promoting disassembly of THO and consequent R loop formation. Compared to LNA, sen1-1 recombination levels increased 2-fold in LNAT. This demonstrates for sen1-1 in contrast to THO mutants, that RNA cleavage in the context of a pA is not sufficient to relieve recombination. To reiterate this point, Figure S2 shows that in a ribozyme containing substrate, recombination levels in sen1-1 are reduced similar to hpr1Δ. As both ribozyme cleaved ends are unprotected they are likely to be degraded and so reduce R loop forming substrate. However, this appears not to be the case if RNA in sen1-1 cells is cleaved at a pA, possibly as RNA downstream to the polyA signal may be less packaged and so temporarily protected from degradation by R loop formation. Although these studies require a more detailed biochemical analysis, we predict from these initial results that R loops may play a role in transcriptional termination.

26In summary the molecular and genetic effects of Sen1 inactivation presented here reveal that Sen1 acts to protect the heavily transcribed genome from R loop-mediated DNA damage. Of note, mutations in the helicase domain of the human SEN1 gene ortholog SETX (encoding Senataxin) cause the neurodegenerative diseases, Ataxia with Oculomotor Apraxia Type II (AOAII), and juvenile amyotrophic lateral sclerosis (ALS4). Like sen1-1 these SETX mutants show defects in transcription, RNA processing, and DNA damage repair. It remains to be established whether the tendency of transcription to induce R loop formation is a general feature of all eukaryotic genomes. It is possible that a range of dedicated helicases act to resolve these potentially harmful structures.

27Yeast strains, plasmids, and primers are listed in Tables S1–S3. Standard methods are detailed in the Supplemental Experimental Procedures. In outline, genetic crossing of single mutants (sen1-1 and nrd1-102) with HR and NHEJ mutants employed standard conditions. Recombination frequencies were scored by counting of LEU+ cells or by FACS analysis of GFP+ cells. GFP-Rad52 nuclear foci were detected by epifluorescent microscopy.

28The transcription run on (TRO) method and probes for pKGG are as described.

29For RNA isolation, strains were grown in minimal (selective) media at indicated temperatures. After acidic phenol RNA isolation, RNA (15 μg) was separated by 1% formaldehyde agarose gel electrophoresis. RNA immobilized on nitrocellulose membranes was detected with random primed probes.

30ChIP employed real-time qPCR as previously described. S9.6 purified antibody was employed for immunoprecipitation and was a kind gift from Stephen Leppla and Antonin Morillon (Institut Curie, Paris). For DIP analysis, sonicated, deproteinized chromatin was immunoprecipitated with S9.6 antibody and amplified by qPCR as further detailed in the Supplemental Experimental Procedures. RNase H sensitivity was measured by treatment with RNase H prior to immunoprecipitation.

31Sequential RNase H (NEB, 2 hr at 37°C) and DNase I (Roche, 4 hr at 37°C) digestion of genomic DNA (5 μg) isolated from logarithmic phase cells cultivated for 1 hr at 37°C with yeast breaking buffer (2% [v/v] Triton X-100, 1% [w/v] SDS, 100 mM NaCl, 10 mM Tris [pH 8.0], and 1 mM EDTA [pH 8.0]), phenol, and glass beads. Isolated RNA was reverse transcribed (Invitrogen Superscript III) according to the manufacturer.

32These are presented in the Supplemental Experimental Procedures and include in vitro 3′ end processing, 3′end RACE, and microarray analysis.