Yeast Sen1 Helicase Protects the Genome
from Transcription-Associated Instability
Hannah E. Mischo,1Bele ´n Go ´mez-Gonza ´lez,2Pawel Grzechnik,1Ana G. Rondo ´n,1,2Wu Wei,3Lars Steinmetz,3
Andre ´s Aguilera,2,* and Nick J. Proudfoot1,*
1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
2Centro Andaluz de Biologia Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Avenida Americo Vespucio s/n,
Sevilla 41092, Spain
3European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
*Correspondence: firstname.lastname@example.org (A.A.), email@example.com (N.J.P.)
Sen1 of S. cerevisiae is a known component of the
NRD complex implicated in transcription termination
of nonpolyadenylated as well as some polyadeny-
lated RNA polymerase II transcripts. We now show
that Sen1 helicase possesses a wider function
by restricting the occurrence of RNA:DNA hybrids
that may naturally form during transcription, when
into RNA protein complexes. These hybrids displace
the nontranscribed strand and create R loop struc-
tures. Loss of Sen1 results in transient R loop accu-
mulation and so elicits transcription-associated
recombination. SEN1 genetically interacts with DNA
repair genes, suggesting that R loop resolution
requires proteins involved in homologous recombi-
nation. Based on these findings, we propose that
R loop formation is a frequent event during transcrip-
tion and a key function of Sen1 is to prevent their
accumulation and associated genome instability.
(Pol II) on protein-coding genes are immediately processed,
packaged, and exported to the cytoplasm (Luna et al., 2008;
Moore and Proudfoot, 2009). Messenger RNA (mRNA) pack-
aging protects transcripts from degradation, but also the DNA
template from invasion of nascent RNA into the DNA duplex
behind elongating Pol II (Aguilera and Go ´mez-Gonza ´lez, 2008).
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-associatedmutation (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 (Cha ´vez
et al., 2000; Fischer et al., 2002; Gallardo and Aguilera, 2001;
Gonza ´lez-Aguilera et al., 2008; Huertas and Aguilera, 2003; Li
and Manley, 2005). R loop formation in these mutants may also
be connected to Pol II stalling, consequently interfering with
processive elongation (Mason and Struhl, 2005; Rondo ´n et al.,
2003) and RNA processing (Libri et al., 2002; Rougemaille
et al., 2008). Similarly, DNA replication may be compromised
when replication forks encounter R loops or a stalled Pol II (Well-
inger et al., 2006).
Although 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 (Yu
et al., 2003). Both processes are initiated by activation induced
deaminase (AID) (Muramatsu et al., 1999). Double-strand breaks
(DSBs) subsequently trigger CSR via nonhomologous end
joining (NHEJ) (Yu and Lieber, 2003). 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 (Go ´mez-Gonza ´lez and Aguilera, 2007; Gon-
za ´lez-Aguilera et al., 2008).
Many events during transcription are orchestrated by proteins
binding tothe carboxy-terminal domain (CTD) ofthe PolIIlargest
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 (Kim et al.,
2009; Komarnitsky et al., 2000). 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 polyadeny-
lation complex (CF IA/B and CPF), as well as degradation of the
Kim et al., 2004b; Meinhart and Cramer, 2004).
Termination of many noncoding RNAs requires an additional
component, the NRD complex (Sen1, Nab3, and Nrd1), in
which Nrd1 is bound to ser5-P CTD (Steinmetz et al., 2001;
Vasiljeva et al., 2008). NRD-dependent termination also
requires recognition of frequent short RNA sequences by Nrd1
Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc. 21
Sen1 Removes R Loops during Transcription
22 Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc.
may vary for different terminators (Kuehner and Brow, 2008).
Nrd1 ser5-P CTD specificity confines this termination pathway
promoter distal Nrd1/Nab3 binding sites unrecognized (Arigo
et al., 2006a; Gudipati et al., 2008). 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) (Arigo et al., 2006b; Thiebaut et al., 2006; Vasiljeva and
Buratowski, 2006). 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 (Neil et al., 2009;
Xu et al., 2009). Importantly, both termination pathways can
substitute for each other and so provide mutual fail-safe
termination mechanisms (Kim et al., 2006; Rasmussen and
Culbertson, 1998). 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 (Rondo ´n et al.,
SEN1 codes for a 240 kDa superfamily I helicase (DeMarini
et al., 1992), and its S. pombe homolog possesses 30-50nucleic
acid unwinding activity (Kim et al., 1999). 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 (Nedea et al., 2008;
Ursic et al., 1995; Winey and Culbertson, 1988). 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 (Ursic et al.,
2004). 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 (Kawauchi et al.,
2008; Rasmussen and Culbertson, 1998; Steinmetz et al.,
2001, 2006; Ursic et al., 1997). 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) (Ghaem-
maghami et al., 2003), 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.
Role of Sen1 Helicase Domain in Transcription
Mutation of the Sen1 helicase domain results in genome-wide
transcription termination defects of noncoding RNAs, but also
of some protein coding genes (Steinmetz et al., 2006). 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 (Morillon et al.,
2003), sen1-1 mutants grown for 150 min at nonpermissive
temperature (37?C) show a strong termination defect (Figure 1A,
upper panels) (Rondo ´n et al., 2009). 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.
To 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
C-terminal helicase domain [Sen1(1212)] (Nedea et al., 2008).
As shown in Figure 1A, Sen1(1212) but not Sen1(323) rescued
the sen1-1 termination defect, implying that the sen1-1 termina-
tion 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 (Kawauchi et al., 2008).
The above results indicate that the Sen1 helicase domain is
required both for efficient Pol II termination and mRNA accumu-
lation. As these effects could be attributed to defective 30end
processing, we employed an in vitro cleavage and polyadenyla-
tion assay using CYC1 30flanking RNA as the pA substrate
(Figure 1C). sen1-1 shows no defects in RNA 30end 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 30
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 30end formation
but stabilized the 30end cleavage product, indicative of loss
of exonuclease ‘‘torpedo’’ function (Kim et al., 2004b; Luo
Figure 1. Sen1 Helicase Is Required for Transcription Termination but Not Transcript 30Processing
empty vector, Sen1(323), or Sen1(1212) constructs. Transformants were grown for 150 min at 37?C before TRO. Bottom: quantification based on four repeat exper-
(C) In vitro cleavage and polyadenylation assays performed with extracts from WT and mutant cells grown for 150 or 90 min (rna14-1) at 37?C with CYC1 30pA as
substrate. Positions of uncleaved, polyadenylated, cleaved, and 30end cleavage product are indicated.
All error bars represent the standard deviation (SD). See also Figure S1.
Sen1 Removes R Loops during Transcription
Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc. 23
et al., 2006). Overall, these combined analyses show that the
Sen1 helicase is dispensable for 30transcript processing but is
required to promote transcriptional termination.
Mitotic Recombination Is Increased in sen1-1
Since S. pombe Sen1 can use RNA:DNA hybrids as an in vitro
substrate (Kim et al., 1999), 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 (Huertas and Aguilera, 2003).
RNA:DNA hybrids may also be naturally encountered in tran-
scribed regions downstream of pA signals, where THO is unde-
tectable on chromatin (Kim et al., 2004a; Luna et al., 2005). We
therefore tested whether sequences downstream of a pA signal
elicit TAR in sen1-1. We employed a plasmid borne recombina-
tion substrate that carries two truncated regions of LEU2 over-
lapping by 600 nt of homologous sequence (LNA). Lack of
THO elicits TAR in LNA and consequent restoration of LEU2,
as previously shown (Figure 2A) (Prado et al., 1997). In contrast,
when transcription between both repeats is terminated by inser-
tion of the CYC1 38nt pA signal (CYC1t, LNAT), recombination
Similar analysis of LNA and LNAT transformed rat1-1 and
rna14-1 showed no detectible increase in recombination, con-
firming that defects in CPF/Rat1 dependent transcription termi-
nation per se do not promote recombination (Luna et al., 2005).
Figure 2. sen1-1 Are Hyperrecombinogenic
(A) Recombinationsubstrates LNAand LNAT. Transformantswere grown for3–4 daysat 30?C.Recombinationgenerates afunctional LEU2,allowingselection of
recombinants on leu-deficient plates. Quantification of recombinants formed from six colonies of four to six transformants is presented.
(B) Recombinants formed in WT and sen1-1 transformed with L and LYDNS containing homologous repeats separated by 39 or 3900 nt.
(C) As in (B), with the LLacZ and LPHO5 substrates under control of either LEU2 or glucose-repressed GAL1 promoters to stimulate high or low expression.
(D) Diagram and recombination quantification of chromosomal construct crossed into the WT and sen1-1.
All error bars represent the SD. See also Figure S2.
Sen1 Removes R Loops during Transcription
24 Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc.
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 irrespec-
tive of the CYC1t. This lack of CYC1t suppression reiterates the
sen1-1 CYC1 termination defect previously reported (Kawauchi
et al., 2008; Steinmetz et al., 2006). Moreover, the fact that
CYC1t (in LNAT) further stimulated recombination may reflect
an increase in R loop formation downstream of pA signals.
To determine whether this recombination phenotype was
specific to Sen1, we similarly tested other NRD complex compo-
nents. 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 biogen-
esis. The fact that CYC1t abrogates recombination in NRD
mutants but stimulates recombination in sen1-1 clearly sepa-
rates Sen1 function from Nab3 and Nrd1 and argues that Sen1
plays a distinct role outside the NRD complex.
sen1-1 Hyperrecombination Depends on Transcription
Hyperrecombination 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 (Gonza ´lez-Aguilera et al.,
2008; Huertas and Aguilera, 2003). To test whether sen1-1
shows a similar transcription-dependent recombination pheno-
type, 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.
RNA:DNA Hybrids Form in sen1-1
Evidence 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 (Go ´mez-Gonza ´lez
and Aguilera, 2007). Employing 50and 30truncated 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.
AID C to U deamination preferentially occurs in a WRC (or
GYW on the opposite strand) sequence (Pham et al., 2003).
In regions of R loop formation, AID has access to the nontran-
scribed strand (NTS), although some mutations occur on the
transcribed strand (TS) (Go ´mez-Gonza ´lez and Aguilera, 2007).
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, Fig-
ure 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 transform-
ants 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 (Go ´mez-Gonza ´lez and Aguilera, 2007). 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
We 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 (Gonza ´lez-Aguilera et al., 2008;
Huertas and Aguilera, 2003). 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 mft1D,
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
mft1D 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
To 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) (Hu et al.,
2006). 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
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,
Sen1 Removes R Loops during Transcription
Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc. 25
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
hpr1D gave significant but lower levels of hybrid signal over
the 50positioned LEU sequence (LGZ2) but not over LGZ5 (Fig-
ure 4A). The 30LGZ5 probe spans the LEU2 pA signal and so will
lack transcripts in hpr1D due to Pol II termination. sen1-1 in
contrast istermination defective sothatread-through transcripts
in this strain will still elicit R loop formation.
The S9.6 antibody was also employed in a regular ChIP anal-
ysis 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 50end in WT and sen1-1 cells,
signals were detected wherever Pol II was present on chro-
matin (Kawauchi et al., 2008). Of note, in chromatin isolated
from rna14-1 that shows no recombination phenotype, little
Figure 3. sen1-1 Cells Form a Substrate for AID and RNase H
(A) AID overexpression increases sen1-1 TAR. pGLG recombinants forming GFP were counted after 12–16 hr growth at 30?C by FACS. AID coexpression
increases GFP-positive cells in both WT and sen1-1 strains (p = 0.018, Wilcoxon test).
(B) Coexpression of AID and pLAUR-induced mutations within URA3 in pLAUR were scored as 5-FOA resistant.
(C)URA3sequence frommutantswasamplified andsequenced.Thefrequencyofpoint mutationsoneitherstrandisdepictedgraphically(seeFigureS3formore
(D) Effect of galactose-induced expression from plasmids pRNH201 (coding RNase H RNH201), pYsen1 (aa 1281–2231 of Sen1 cloned into pYES2), or pYES2
alone on the recombination frequencies in WT and sen1-1 cells produced by the LLacZ system. Note that double selection and growth on galactose reduces the
sen1-1 viability and therefore recombination frequencies as compared to data in Figure 2C.
(E) Recombination frequency analysis in the THO-complex mutant mft1D with the GLG recombination substrate and AID to increase recombination rates. Over-
expression of Sen1 helicase reduces recombination frequency.
All error bars represent the SD. See also Figure S3.
Sen1 Removes R Loops during Transcription
26 Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc.
hybrid could be detected downstream of the pA signals, even
through Pol II is still present at these positions. This may indi-
cate 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 indi-
cate a region that is particularly prone to form RNA:DNA
In 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 frac-
tionation 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 down-
stream of the PMA1 pA signal. This suggests that in sen1-1
a higher fraction of RNA forms R loop structures with genomic
downstream of the pA site.
From 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
Figure 4. R Loops Form in sen1-1 Cells
(A) DIP analysis on pGLLacZ in sen1-1, hpr1D, and WT
cells with antibody against RNA:DNA hybrids (S9.6).
Coimmunoprecipitated DNA was detected by real-time
qPCR. Inocules were grown in raffinose, induced with
galactose for 1 hr at 37?C, and successively repressed
at 37?C by addition of 4% glucose.
(B) Reverse transcription (RT) of RNA isolated from
genomic DNA preparations after treatment with RNase
H. The levels of RNase H resistant RNA were measured
after RT with P5 and P7 primers followed by PCR with
P5 amplicon (as shown on the gene map). Signals
obtained were free from DNA contamination based
on minus RT controls. Left: representative gel. Right:
quantification of RT normalized triplicate repeats by real-
time qRT-PCR, further normalized to P5 amplicon signal
obtained from non-RNase H-digested samples.
All error bars represent the SD. See also Figure S4.
throughout the transcribed PMA1 gene, with
a particular prevalence over the pA site.
SEN1 Genetic Interaction with
Homologous Recombination Genes
Experiments 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 compar-
ison 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) (David
et al., 2006). This revealed that among the transcripts whose
expression was significantly changed (adjusted p value of <
0.01), stable unannotated transcripts were overrepresented
of these genes (Figure S5A and Table S4). Importantly, THO or
THSC genes were absent among the 347 ORFs that were signif-
icantly 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
To define which DNA repair mechanism was induced by R
loops formed in sen1-1, we searched for synthetic genetic inter-
actions 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
Sen1 Removes R Loops during Transcription
Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc. 27
deletion mutants, both found in a complex with Xrs2 and
involved in initial recognition and ss resection at a DSB (Fig-
ure 5A). The critical need for HR in sen1-1 cells is demonstrated
by the phenotypes of double mutants of sen1-1 and rad52D,
sgs1D, srs2D, or mus81D. These all showed synthetic defects
or displayed increased sensitivity to growth in hydroxyurea
(HU). Incontrast, sen1-1yku70Ddoublemutants showedneither
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.
DNA Damage Foci in sen1-1 Nuclei
In cells that accumulate DNA damage, factors involved in DNA
repair are rapidly recruited to the damage site (Lisby et al.,
2001). 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; Fig-
ure 6A). This percentage of sen1-1 cells forming foci increased
from 8% at 25?C to 13% when cultures were shifted to semiper-
missive (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
(Rasmussen and Culbertson, 1998). 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 pheno-
types observed in this work and support the assumption that
they are caused by mutation of Sen1 helicase domain in sen1-1.
Using this assay as an indicator of ongoing HR in sen1-1, we
transcription dependent R loops. WT and sen1-1 transformed
with Rad52-GFP and pYSen1, which encodes the Sen1 helicase
Figure 5. sen1-1 Shows Synthetic Genetic
Interaction with DNA Damage Repair Genes
(A) Synthetic interactions between sen1-1 and
MRX gene mutations: mre11D, rad50D. Also
shown are synthetic interactions between sen1-1
and HR gene mutants sgs1D and rad52D. White
boxes indicate spores that carry both mutations.
(B) Analysis of HU sensitivity of double mutants
grown at 25?C. Growth was compared on YPAD
plates ± 50 mM HU (10 mM * or 100 mM ** as indi-
See also Figure S5 and Table S4.
30?C. After addition of glucose or galac-
tose 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) (Ursic et al., 2004) (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
diately repaired or the cells die.
The deleterious effects of transcription on genome integrity
have been suggested by various observations (Aguilera, 2002).
aging, and export in eukaryotes is disrupted, genome instability
can be observed (Baaklini et al., 2004; Broccoli et al., 2004;
Jimeno et al., 2002; Luna et al., 2005). 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 recombi-
nation. A large fraction of this instability is transcription depen-
dent 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 adele-
terious 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 pres-
ence 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 Removes R Loops during Transcription
28 Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc.
We suggest that as soon as the nascent transcript emerges
from the polymerase body, mRNA packaging and R loop forma-
tion occur in kinetic competition (Figure 7). A fragile equilibrium
between protective mRNA packaging and the hiding of specific
recognition sequences is likely to exist (Bucheli and Buratowski,
2005; Bucheli et al., 2007). Therefore, RNA packaging is likely to
be incomplete so leaving some transcript available for R loop
on NRD and by inference on Sen1 (Arigo et al., 2006b). If R loops
formed in sen1-1 extend to many CUT loci, then their accumula-
tion, even if transient, would cover substantial regions of the
genome. In both CUTs and mRNA coding genes, R loops could
Figure 6. DNA Repair Foci in sen1-1 Nuclei
(A) Time course of WT and sen1-1 grown at log phase and shifted to 30?or 37?C. At indicated time points aliquots were spotted on microscope slides and foci-
containing cells scored based on 300 cells. Representative pictures (top left) and quantification of 3-5 repeats are shown (bottom left). Simultaneously isolated
RNA was analyzed by Northern Blot against SNR13 to show accumulation of bi-cistronic SNR13-TRS31 transcript in sen1-1.
(B) WT and sen1-1 cells transformed with pWJ144 and pYSen1 grown in raffinose and shifted to 30?C for 1hr, before Sen1 helicase fragment expression was
induced or repressed by addition of 2% galactose or 2% glucose to the medium respectively. Only WT in galactose shown and foci containing cells scored
as in A.
(C) sen1-1rpb1-1 cells transformed withpWJ144were grown inlogarithmic phaseat25?C and thenshifted to37?C.Shutoff of transcription resultsindecreaseof
Rad52 foci, either by reduced accumulation or Rad52 turnover.
All error bars represent the SD.
Sen1 Removes R Loops during Transcription
Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc. 29
interfere with DNA replication, induce ssDNA breaks, or be
recognized as recombination intermediates. Any of these possi-
bilities 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 (Go ´mez-Gonza ´-
lez et al., 2009). Alternatively, these differences may hint at a
transcription-independent role of Sen1 in DNA damage repair
that is yet to be uncovered.
How may R loop accumulation in sen1-1 be related to its tran-
scription termination defect? R loops were originally hypothe-
sized to slow down transcription elongation, thereby enhancing
termination (Proudfoot, 1989). This would give time for the
Rat1 50-30exonuclease ‘‘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 (Huertas and Aguilera, 2003; Mason
and Struhl, 2005). Employing sen1-1, in which transcript pro-
cessing is normal, we predict that reduced steady-state RNA
accumulation is due to reduced transcript elongation. Further-
more, 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 (Kawauchi
et al., 2008), it should serve as a 30processing signal (Figure 1C),
promoting disassembly of THO and consequent R loop forma-
tion (Kimet al., 2004a). 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 hpr1D
(Huertas and Aguilera, 2003). 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
Figure 7. Cotranscriptional Functions of Sen1
(A) Model for Sen1 cotranscriptional function especially in
(B)Model for Rloop accumulation insen1-1 showing three
ways they may elicit HR: processing of nicks in ssDNA,
ssDNA recognition, and collapse of colliding replication
case if RNA in sen1-1 cells is cleaved at a pA,
possibly as RNA downstream to the polyA
protected from degradation by R loop forma-
tion. 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.
In summary the molecular and genetic effects
of Sen1 inactivation presented here reveal that
Sen1 acts to protect the heavily transcribed
genomefrom Rloop-mediated DNAdamage. 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 (Moreira et al., 2004; Suraweera et al.,
2007, 2009). It remains to be established whether the tendency
of transcription to induce R loop formation is a general feature
ofall eukaryotic genomes.Itispossible thata rangeofdedicated
helicases act to resolve these potentially harmful structures.
Yeast Cultivation and Genetic and Cell Biology Methods
Yeast strains, plasmids, and primers are listed in Tables S1–S3. Standard
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.
Transcription Run On Analysis
The transcription run on (TRO) method and probes for pKGG are as described
(Morillon et al., 2003; Rondo ´n et al., 2009).
Northern Blot Analysis
For RNA isolation, strains were grown in minimal (selective) media at indicated
temperatures. After acidic phenol RNA isolation, RNA (15 mg) was separated
by 1% formaldehyde agarose gel electrophoresis. RNA immobilized on nitro-
cellulose membranes was detected with random primed probes.
Chromatin and DNA Immunoprecipitation
ChIP employed real-time qPCR as previously described (Rondo ´n et al., 2009).
S9.6 purified antibody was employed for immunoprecipitation and was
a kind gift from Stephen Leppla (Hu et al., 2006) 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 (Liu et al., 2005).
RNase H sensitivity was measured by treatment with RNase H prior to
Sen1 Removes R Loops during Transcription
30 Molecular Cell 41, 21–32, January 7, 2011 ª2011 Elsevier Inc.
RT Analysis of Genomic DNA-Associated RNA
Sequential RNase H (NEB, 2 hr at 37?C) and DNase I (Roche, 4 hr at 37?C)
digestion of genomic DNA (5 mg) 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 (Invitro-
gen Superscript III) according to the manufacturer.
Further Standard Experimental Procedures
These are presented in the Supplemental Experimental Procedures and
include in vitro 30end processing, 30end RACE, and microarray analysis.
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and four tables and can be found with this article online at
Thanks to our lab colleagues, especially Monika Gullerova and Jurgi Camb-
long, Julien Gagneur, Sandra Clauder-Muenster, and Charles Giradot
(submission to ArrayExpress). Funding was from the Wellcome Trust to
N.J.P., the Spanish Ministry of Science and Innovation (BFU2006-05260 and
Consolider Ingenio 2010 CSD2007-0015) and Junta de Andalucı ´a (BIO102
and CV2549) to A.A., and a joint ESF grant to N.J.P. and A.A. (NuRNASu).
H.E.M. was recipient of a B.I.F. studentship and a European Molecular Biology
Organization (EMBO) short-term fellowship. B.G. received a predoctoral FPU
training grant from the Spanish Ministry of Science and Innovation. P.G. was
supported by a long-term EMBO fellowship.
Received: February 23, 2010
Revised: September 21, 2010
Accepted: October 21, 2010
Published: January 6, 2011
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