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Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
A Role for Small RNAs in DNADouble-Strand Break RepairWei Wei,1,2,9 Zhaoqing Ba,2,3,9 Min Gao,4 Yang Wu,2 Yanting Ma,2 Simon Amiard,5 Charles I. White,5
Jannie Michaela Rendtlew Danielsen,4,6 Yun-Gui Yang,4 and Yijun Qi2,7,8,*1Graduate Program, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China2National Institute of Biological Sciences, Number 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China3College of Life Sciences, Beijing Normal University, Beijing 100875, China4Genome Structure and Stability Group, Disease Genomics and Individualized Medicine Laboratory, Beijing Institute of Genomics,
Chinese Academy of Sciences, Number 7 Beitucheng West Road, Chaoyang District, Beijing 100029, China5Genetique, Reproduction et Developpement, Unite Mixte de Recherche Centre National de la Recherche Scientifique 6247,
Clermont Universite, Institut National de la Sante et de la Recherche Medicale U931, Aubiere 63177, France6The Novo Nordisk Foundation Center for Protein Research, Ubiquitin Signalling Group, Faculty of Health Sciences, Blegdamsvej 3b,
2200 Copenhagen, Denmark7Tsinghua-Peking Center for Life Sciences, Beijing 100084, China8School of Life Sciences, Tsinghua University, Beijing 100084, China9These authors contributed equally to this work
*Correspondence: qiyijun@nibs.ac.cn
DOI 10.1016/j.cell.2012.03.002
SUMMARY
Eukaryotes have evolved complex mechanisms torepair DNA double-strand breaks (DSBs) throughcoordinated actions of protein sensors, transducers,and effectors. Here we show that �21-nucleotidesmall RNAs are produced from the sequences inthe vicinity of DSB sites in Arabidopsis and in humancells. We refer to these as diRNAs for DSB-inducedsmall RNAs. In Arabidopsis, the biogenesis of diR-NAs requires the PI3 kinase ATR, RNA polymeraseIV (Pol IV), and Dicer-like proteins. Mutations in theseproteins as well as in Pol V cause significant reduc-tion in DSB repair efficiency. In Arabidopsis, diRNAsare recruited by Argonaute 2 (AGO2) to mediate DSBrepair. Knock down of Dicer or Ago2 in human cellsreduces DSB repair. Our findings reveal a conservedfunction for small RNAs in the DSB repair pathway.We propose that diRNAs may function as guidemolecules directing chromatin modifications or therecruitment of protein complexes to DSB sites tofacilitate repair.
INTRODUCTION
DNA double-strand breaks (DSBs) are deleterious forms of DNA
damage that can cause mutations, genome instability, and cell
death. Efficient repair of DSBs is thus critical for themaintenance
of genome integrity and cell survival. DSBs are mainly repaired
by nonhomologous end joining (NHEJ) or homologous recombi-
nation (HR) (Ciccia and Elledge, 2010; Lieber, 2010; Moynahan
and Jasin, 2010; Puchta, 2005; San Filippo et al., 2008; Sasaki
et al., 2010). NHEJ repairs DSBs in an efficient but error-prone
fashion. It can cause deletions or insertions at the break site
because of the modification of DNA ends before joining (Lieber,
2010). In contrast, HR is considered error free, but it requires
resection of the DSB and a sister chromatid as template for
repair (Moynahan and Jasin, 2010; San Filippo et al., 2008; Sa-
saki et al., 2010). Single-strand annealing (SSA) is a particular
type of HR that takes place when DSB resection occurs at repet-
itive sequences, providing complementary single strands that
can then anneal (Ciccia and Elledge, 2010; Hartlerode and
Scully, 2009). These repair pathways all require well-regulated
and coordinated enzymatic actions of protein sensors, trans-
ducers, and effectors in the DSB signaling cascade (Ciccia and
Elledge, 2010; Huen and Chen, 2008; Polo and Jackson, 2011).
Small RNAs have emerged as key players in various aspects of
biology. Three major classes of small RNAs have been discov-
ered in eukaryotes: microRNAs (miRNAs), small interfering
RNAs (siRNAs), and Piwi-interacting RNAs (piRNAs). miRNAs
and siRNAs are processed by RNase III domain-containing Dicer
or Dicer-like (DCL) proteins from their double-stranded RNA
(dsRNA) precursors. Via association with members of the Argo-
naute (AGO) family, small RNAs mediate transcriptional or post-
transcriptional regulation of gene expression (Baulcombe, 2004;
Carthew and Sontheimer, 2009). AGO proteins contain several
characteristic domains: a variable N-terminal domain and con-
served PAZ, MID, and PIWI domains (Tolia and Joshua-Tor,
2007). The PAZ and PIWI domains bind to the 30 and 50 ends of
small RNAs, respectively (Ma et al., 2004; Ma et al., 2005), and
the PIWI domain has endonuclease activity when an Asp-Asp-
His catalytic triad is present (Rivas et al., 2005; Song et al., 2004).
In plants, the most abundant small RNAs are heterochromatic
siRNAs (hc-siRNAs) derived from transposons and other repeti-
tive sequences. Single-stranded RNA transcripts are presum-
ably generated from DNA repeats by DNA-dependent RNA
polymerase IV (Pol IV) (Herr et al., 2005; Onodera et al., 2005)
and converted by RNA-dependent RNA polymerase 2 (RDR2)
Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc. 1
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
into dsRNAs (Xie et al., 2004), which are in turn processed
by DCL3 into 24 nucleotide (nt) hc-siRNAs (Xie et al., 2004).
Hc-siRNAs are associated with AGO4 subfamily members and
direct DNA methylation by the de novo DNA methyltransferase
DRM2 through a pathway known as RNA-directed DNA methyl-
ation (RdDM) (Law and Jacobsen, 2010; Matzke et al., 2009).
Functionally analogous to plant hc-siRNAs, piRNAs are specific
to animals and most often specifically generated in the germ line
to inactivate transposons (Malone and Hannon, 2009). In addi-
tion to these three classes of small RNAs, other types of small
RNAs have also been found in various organisms. Tetrahymera
thermophila contains scan RNAs (scnRNAs) that are involved
in developmentally regulated DNA elimination (Mochizuki and
Gorovsky, 2004; Yao and Chao, 2005). More recently, high-
throughput approaches have identified promoter-associated
short RNAs (PASRs) (Kapranov et al., 2007), termini-associated
short RNAs (TASRs) (Kapranov et al., 2007; Kapranov et al.,
2010), and transcription-initiation RNAs (tiRNAs) (Taft et al.,
2009) in animals.
In light of the expanding universe of small RNAs and their
increasingly diverse biological roles, we explored whether small
RNAs could play a role in DSB repair. Using well-established
reporter assays for DSB repair in Arabidopsis thaliana and
human cells, we found that DSBs trigger the production of small
RNAs from the sequences in the vicinity of DSB sites and these
small RNAs are required for efficient DSB repair. Our results
reveal an unsuspected and conserved role for small RNAs in
the DSB repair pathway.
RESULTS
DCLs Are Required for Efficient DSB Repairin Arabidopsis
We used an Arabidopsis transgenic line carrying the DGU.US
reporter in which expression of the endonuclease I-SceI,
induced by crossing, introduces a single DSB in the genome
(Mannuss et al., 2010; Orel et al., 2003). Repair of the DSB
through SSA mechanism restores b-glucuronidase (GUS)
expression, which provides a visible and quantitative readout
of DSB repair events (Figure 1A). Confirming published results
(Mannuss et al., 2010; Orel et al., 2003), crossing the DGU.US
reporter (R) line with a DSB-triggering (T) line that expresses
I-SceI induced DSB repair with a repair rate about two orders
of magnitude higher than that of uncrossed R line (Figures 1B,
1D, and 1E). We then introduced, via crossing, this reporter
system into an Arabidopsis atr mutant background to assay
the DSB repair rate in this mutant (Figure S1, available online).
ATR encodes a phosphatidyl inositol 3-kinase-like protein kinase
(PI3 kinase) that primarily responds to stalled replication forks
and also acts with ATM (Ataxia Telangiectasia Mutated, another
PI3 kinase) in DSB response (Amiard et al., 2010; Culligan et al.,
2004; Culligan and Britt, 2008; Culligan et al., 2006; Jazayeri
et al., 2006). We found that the repair efficiency was greatly
reduced in the atr mutant as determined by GUS staining or
PCR detection of repaired DNA (Figures 1B, 1D, and 1E).
DCLs are required for small RNA biogenesis in Arabidopsis
(Xie et al., 2004). We next examined whether DCLs are involved
in DSB repair. We introduced the GUS reporter system into dcl2,
2 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
dcl3, and dcl4mutant backgrounds. Intriguingly, compared with
those in the wild-type controls, the repair rates were reduced by
42%, 90%, and 44% in the dcl2, dcl3, and dcl4 mutant back-
grounds, respectively (Figures 1C, 1D, and 1E), suggesting that
DCL2, DCL3, and DCL4 play partially redundant roles in DSB
repair and that DCL3 is the major player.
DSBs Trigger Production of Small RNAs fromSequencesFlanking the DSB SitesThe requirement of DCLs for efficient DSB repair suggested
the involvement of small RNAs in this process. Thus, we sought
to examine whether small RNAs were produced from the
sequences around the DSB site by northern blot analysis. When
a probe that recognizes the �450 nt sequence flanking the
I-SceI site (Figure 1A) was used, small RNAs were barely detect-
able in theR or T line but were readily detected in the progenies of
RxT cross (Figure 2A). These small RNAswere nameddiRNAs for
DSB-induced small RNAs. Deep sequencing analyses revealed
that diRNAs were specifically derived from both sense and anti-
sense strands of the sequence around the I-SceI site (Figure 2B).
Confirming thenorthernblot results,�40 timesmorediRNA reads
were obtained from the RxT sample compared to those from the
uncrossed R line (Figure 2B), whereas the expression profiles of
endogenous small RNAs (miRNAs and siRNAs) were comparable
in these two samples (Table S1). Moreover, we found that the
production of diRNAs was dependent on ATR. In the atr mutant
background, the diRNAs were barely detectable either by
northern blot (Figure 2C) or deep sequencing (Table S2).
We extended the diRNA analysis to another DSB repair
reporter (DU.GUS) system (Orel et al., 2003). In this system,
the DSB site generated by I-SceI digestion is repaired by the
HR pathway that involves gene conversion through synthesis-
dependent strand annealing (SDSA). We detected the produc-
tion of diRNAs in the DU.GUSxT plants by deep sequencing
(Figure 2D). The amount of diRNAs produced in the DU.GUSxT
plants was approximately one-fifth of that detected in the RxT
plants (Figures 2B and 2D). This is in correlation with the lower
DSB repair frequency in the DU.GUS line, which has been
reported to be about five times lower than that in the DGU.US
line (Orel et al., 2003).Taken together, these observations
demonstrate that DSBs induce the production of diRNAs specif-
ically around the DSB sites.
diRNAs Play a Role in DSB RepairTo investigate the role of diRNAs in DSB repair, we first examined
the production of diRNAs in the dcl mutants that had reduced
repair rates (Figures 1C, 1D, and 1E). Compared to the wild-
type controls, dcl2, dcl3, and dcl4 accumulated many fewer
diRNAs (Figure 3A and Table S2). In particular, the production
of diRNAs was reduced by 98% in the dcl3 mutant, consistent
with its predominant effect on the repair efficiency (Figures 1C,
1D, and 1E). DCL2, DCL3, and DCL4 are known to process
dsRNAs into 22 nt, 24 nt, and 21 nt siRNAs, respectively (Qi
et al., 2005; Xie et al., 2004). It is intriguing that the production
of both 21 nt and 24 nt siRNAs was greatly reduced in each of
the dcl mutants (Figure 3A). This suggests that diRNAs might
be processed from their dsRNA precursors through coordinated
actions of DCL2, DCL3, and DCL4. Coordinated actions of DCLs
Figure 1. DCLs Are Required for Efficient DSB Repair in Arabidopsis
(A) Schematic representation of DSB repair in the DGU.US reporter system. The DGU.US reporter (R) line harbors an I-SceI site located within the direct repeats
(U) of a nonfunctional GUS. Crossing R with the DSB-triggering (T) line that expresses the I-SceI endonuclease introduces a single DSB in the genome of F1
progenies (RxT). Repair of the DSB restores the functional GUS.
(B and C) Representative GUS staining images for DSB repair analysis in the T, R, and RxT plants and RxT plants in the indicated mutant backgrounds (�/�) or
their corresponding wild-type (+/+) backgrounds. See Figure S1 for the crossing scheme.
(D) The relative repair rate in the indicated plants determined by GUS staining. For each genetic background, at least 30 plants from three independent
experiments were stained and blue sectors were counted. The DSB repair rates in the mutant plants (�/�) are presented in relation to those of the corresponding
wild-type (+/+) controls (arbitrarily set to 1.0). Error bars indicate standard error of the mean (SEM), and the asterisks indicate a significant difference between the
indicated groups (t test, p value < 0.001).
(E) Detection of repaired DNA in the indicated plants by PCR using primers p1 and p2 depicted in (A), Histone H4 was also amplified and used as the internal
control. See also Figure S1.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
have been previously shown for the biogenesis of longmiRNAs in
rice (Wu et al., 2010).
The generation of diRNAs from both sense and antisense
strands at approximately equal frequency (Figure 2B) suggested
that they were processed from dsRNA precursors, the produc-
tion of which usually requires RDRs (Xie et al., 2004). RDR2 is
required for the production of hc-siRNAs (Xie et al., 2004),
whereas RDR6 plays a role in the biogenesis of trans-acting
siRNAs (Peragine et al., 2004; Vazquez et al., 2004). We found
that mutations in RDR2 and RDR6 caused 87% and 82%
reductions in diRNA production, respectively (Figure 3A and
Table S2). However, these mutations had no significant effects
on the repair efficiency (Figures 3B and 3C and Figure S3),
which could indicate a redundancy between RDR2 and RDR6
or that other RDRs are involved. Alternatively, these findings
could also imply that there might be a threshold of diRNA
abundance required for its function in DSB repair and that the
levels of diRNAs present in the rdr2 and rdr6 mutants are
sufficient.
We next examined whether Pol IV and Pol V are required for
diRNA biogenesis and DSB repair. Pol IV and Pol V are both
involved in the Arabidopsis RdDM pathway. Pol IV is required
for hc-siRNA biogenesis (Herr et al., 2005; Kanno et al., 2005;
Onodera et al., 2005), whereas Pol V generates nascent scaffold
Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc. 3
Figure 2. DSB Induces the Production of diRNAs Specifically Around the DSB Site in Arabidopsis
(A) Detection of small RNAs in the F1 progenies (RxT), as well as the parental plants (R and T) by northern blot analysis. The probe used for detection of
GUS-derived diRNAs is depicted in Figure 1A. miR173, SIMPLEHAT2 hc-siRNAs, and U6 were probed as controls. nt is an abbreviation for nucleotide.
(B) Deep sequencing analysis of diRNAs generated from DGU.US reporter construct. The y axis represents the number of normalized small RNA reads per
10 million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. The nucleotide
positions (bp) of the components in the DGU.US construct are shown.
(C) Northern blot detection of diRNAs in RxT in Col-0 background (WT), atr mutant (�/�) and its corresponding wild-type (+/+) backgrounds.
(D) Deep sequencing analysis of diRNAs generated from DU.GUS reporter construct. The y axis represents the number of normalized small RNA reads per 10
million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. The nucleotide
positions (bp) of the components in the DU.GUS construct are shown. See Figure S2 for the diagram of DU.GUS reporter construct.
4 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
Figure 3. A Role for diRNAs in DSBRepair in
Arabidopsis
(A) Northern blot analysis of diRNA accumulation
in the indicated mutant (�/�) and the corre-
sponding wild-type (+/+) backgrounds. miR173,
ta-siR255, and SIMPLEHAT2 hc-siRNAswere also
probed and used for verification of respective
mutant backgrounds. U6 was detected and used
as a loading control. nt is an abbreviation for
nucleotide.
(B) The relative DSB repair rates in the indicated
plants determined by GUS staining. For each
genetic background, at least 30 plants from three
independent experiments were stained and blue
sectors were counted. The repair rates in the
mutant plants (�/�) are presented in relation to
those of the corresponding wild-type (+/+)
controls (arbitrarily set to 1.0). Error bars indicate
SEM, and asterisks indicate a significant differ-
ence between the indicated groups (t test,
p value < 0.001).
(C) Detection of repaired DNA in the indicated
plants by PCR.
(D) Detection of diRNAs in the ago4 mutant (�/�)
and the wild-type control (+/+) plants.
(E) The repair rates in the ago4mutant (�/�) and its
corresponding wild-type control (+/+) determined
by GUS staining (upper panel) and PCR (lower
panel).
(F) The repair rates in the drm1/drm2 double
mutant (�/�) and its corresponding wild-type
control (+/+) determined by GUS staining (upper
panel) and PCR (lower panel). See also Figures S3
and S4.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
transcripts upon which RdDM effector complexes are assem-
bled but has no direct role in hc-siRNA biogenesis (Wierzbicki
et al., 2008). Intriguingly, diRNA production was greatly compro-
mised in nrpd1 (NRPD1 encodes the largest subunit of Pol IV) but
increased in nrpe1 (NRPE1 encodes the largest subunit of Pol V)
(Figure 3A and Table S2). Repair rates were reduced by 80% and
50% in the nrpd1 and nrpe1 mutant backgrounds, respectively
(Figures 3B and 3C and Figure S3). These data indicate that
Pol IV and Pol V are involved in DSB repair through regulating
diRNA biogenesis and functioning, respectively.
diRNA-Mediated DSB Repair Does Not Involve the RdDMPathwayDCL3, Pol IV, and Pol V are all components in the RdDMpathway
(Law and Jacobsen, 2010). The dependence of DSB repair on
Cell 149, 1
these genes raised a possibility that
diRNAs function through RdDM to
mediate DSB repair. To test this possi-
bility, we investigated whether AGO4
(the major RdDM effector protein that
binds hc-siRNAs) (Qi et al., 2006; Zilber-
man et al., 2003) and DRM2 (the de
novo DNA methyltransferase that cata-
lyzes RdDM) (Cao and Jacobsen, 2002;
Matzke et al., 2009) are involved in DSB
repair. In the ago4 mutant, the accumulation of diRNAs
was not reduced but instead mildly increased (Figure 3D and
Table S2), and DSB repair efficiency was also not affected (Fig-
ure 3E). Similarly, mutation in DRM1/2 did not have an obvious
effect on DSB repair (Figure 3F).
The reduced DSB repair efficiency observed in dcl3, nrpd1,
and nrpe1 could be caused by dysregulation of genes involved
in DNA damage response in these mutants. To test this possi-
bility, we used quantitative RT-PCR (qRT-PCR) to measure the
expression levels of several genes (MRE11, RAD50, NBS1,
ATM, ATR, RAD51, RPA1, BRCA1, BRCA2, RAD54, RECQ4A,
RAD5A, and RPA2b) that play key roles in DNA damage
response. We found that the expression levels of all the exam-
ined genes were comparable in wild-type and mutant plants
(Figure S4).
–12, March 30, 2012 ª2012 Elsevier Inc. 5
Figure 4. AGO2 Is an Effector Protein of diRNAs
(A and B) AGO2 expression was induced by g-irradiation
as measured at both mRNA and protein levels by qRT-
PCR (A) and western blot (B) analyses, respectively. Error
bar indicates SEM and the asterisk (*) indicates a signifi-
cant difference between the indicated samples (t test, p <
0.001). In (B) tubulin was detected in parallel and used as
a loading control.
(C) Detection of diRNAs in the immunopurified AGO2
complex from the RxT plants by northern blot analysis.
The silver-stained gel shows comparable amounts of
AGO2 complexes were used for RNA extraction.
(D and E) Northern blot (D) and deep sequencing (E)
analyses of diRNAs in the ago2 mutant (�/�) and the
corresponding wild-type (+/+) plants. In (E), reads per
10 million sequences are shown after being normalized
with reads of endogenous miRNAs.
(F) The effect of mutation in AGO2 on the DSB repair rate
was determined by GUS staining. Representative images
are shown in the left. Relative repair rate was calculated
and shown in the right. For each genetic background, at
least 30 plants from three independent experiments were
assayed. Error bars indicate SEM, and the asterisk indi-
cates a significant difference between the indicated
groups (t test, p value < 0.001).
(G) Detection of repaired DNA by PCR in the indicated
plants. See also Figure S5.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
These results argue against the possibility that diRNAs
mediate DSB repair through the RdDM pathway or through the
regulation of genes involved in DSB response.
AGO2 Is an Effector Protein of diRNAsTo dissect the mechanism through which diRNAs act in DSB
repair, we sought to identify the effector protein that recruits
diRNAs. It has been previously reported that the expression of
AGO2 can be induced by g-irradiation, a potent DSB inducer
(Culligan et al., 2006). Consistent with the published results,
we found that expression of AGO2, but not the expression of
other AGOs, was highly induced in plants upon g-irradiation at
both mRNA and protein levels (Figures 4A and 4B and Fig-
ure S5A). This suggested AGO2 as a potential candidate effector
that recruits diRNAs. To test this, we immunopurified AGO2
complexes from the RxT plants as well as the uncrossed R line
(Figure 4C) and examined whether they contained diRNAs.
Northern blot analysis detected diRNAs in the AGO2 complexes
isolated from the RxT plants but not in those from the uncrossed
R line (Figure 4C). Confirming the northern blot results, deep
sequencing analysis revealed that GUS-matching diRNAs
accounted for �3% of the AGO2-bound small RNAs, and there
was a 6.2-fold enrichment of diRNAs in AGO2 relative to those
in the total extracts (Figures S5B and C). In agreement with the
role of AGO2 in recruiting diRNAs, the ago2mutant had dramat-
ically reduced accumulation of diRNAs (Figures 4D and 4E) and
reduced repair rate (Figures 4F and 4G). These results indicate
that AGO2 is an effector protein of diRNAs and plays a role in
DSB repair.
6 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
We reasoned that some diRNAs might be produced from
endogenous loci upon g-irradiation. We immunoprecipitated
AGO2 complexes from plants with or without the treatment of
g-irradiation and analyzed the coimmunoprecipitated small
RNAs by deep sequencing. We identified 150 loci that produced
two times more small RNAs in the g-irradiated plants than in the
unirradiated plants (Table S3). As g-irradiation triggers DSB
randomly in the chromosomes, we were unable to determine
the DSB sites in the g-irradiated plants. It remains to be
confirmed whether these induced small RNAs were DSB
associated.
diRNAs Are Not Involved in the Phosphorylation of H2AXPhosphorylation of histone H2AX, referred to as g-H2AX, is one
of the earliest events in the response to DSBs. g-H2AX plays a
key role in recruiting repair and chromatin remodeling factors
at the sites of DNA damage (Fillingham et al., 2006; Paull et al.,
2000; Podhorecka et al., 2010) and has emerged as a highly
specific and sensitive molecular marker for monitoring DSBs
and their repair (Amiard et al., 2010; Kinner et al., 2008). In
Arabidopsis, phosphorylation of H2AX is dependent on both
ATM and ATR in response to DSBs caused by ionizing radiation,
and ATM has a dominant role (Friesner et al., 2005).
We examined whether diRNAs are required for the phosphor-
ylation of H2AX at DSB sites induced by g-irradiation. We
performed g-H2AX immunofluorescence staining with nuclei
isolated from Arabidopsis leaves. As expected, g-H2AX foci
were not detectable in the nuclei of unirradiated plants. When
plants were irradiated with 25 Gy, 100% of the nuclei from
Figure 5. diRNAs Are Not Involved in the
Phosphorylation of H2AX
(A) Detection of g-H2AX by fluorescent immuno-
staining in the nuclei of wild-type (Col-0) and the
indicated mutant plants without (0 Gy, upper
panels) or with (25 Gy, lower panels) the treatment
of 25 Gy g-irradiation. DNA was stained with DAPI
(blue), and g-H2AX foci were colored in red.
Representative pictures are shown. The scale
bar = 5 mm.
(B) Graphic representation of the number of
g-H2AX foci detected in the unirradiated or irra-
diated wild-type (Col-0) and mutant plants. The
numbers of foci in at least 30 nuclei were counted
for each sample and used to generate the
diagrams. In the right panel, error bars indicate
SEM, and the asterisks (*) indicate a significant
difference between the indicated groups (t test,
p value < 0.001).
See also Figure S6.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
wild-type plants showed g-H2AX foci with a mean of 31 foci per
nucleus (Figures 5A and 5B). The proportion of nuclei showing
g-H2AX foci and the number of foci per nucleus were dramati-
cally reduced in the irradiated atm mutant plants and slightly
but significantly decreased in the irradiated atr mutant plants
(Figures 5A and 5B). However, the amounts of g-H2AX foci in
irradiated nrpd1, dcl3, and ago2mutant plants were comparable
to those in irradiated wild-type plants (Figures 5A and 5B). Immu-
nofluorescence staining with mitotic root tip nuclei showed that
mutations in AGO2 and DCL3 did not decrease the numbers of
g-H2AX foci in M-phase or interphase nuclei (Figure S6). These
data indicate that diRNAs are not involved for the generation of
g-H2AX and diRNAs most likely function downstream of H2AX
phosphorylation.
Cell 149, 1
Detection of diRNAs and Their Rolein DSB Repair in Human CellsOur findings point to a key role for diRNAs
in the DSB repair pathway in plants.
Becausemultiple aspects of this pathway
are highly conserved, we asked whether
diRNAs could also be involved in DSB
repair in mammalian cells. We employed
human U2OS cells carrying a DR-GFP
substrate (DR-GFP/U2OS), which con-
tains two nonfunctional GFP open
reading frames, including one GFP-
coding sequence that is interrupted by
a recognition site for the I-SceI endonu-
clease. Expression of I-SceI leads to
formation of a DSB in the I-SceI GFP
allele, which is repaired by HR using a
nearby GFP lacking N- and C-terminal
GFP-coding sequences, thereby pro-
ducing functional green fluorescent pro-
tein (GFP) that can be readily detected
by flow cytometry (Pierce et al., 1999).
DSBs were induced by the expression
of I-SceI in DR-GFP/U2OS cells (Figure S7). Deep sequencing
analyses demonstrated that, as in Arabidopsis, DSBs in human
cells induced diRNA production from sense and antisense
strands of the sequence close to the DSB site (Figure 6A).
diRNAs appeared to be produced from the vicinity of the DSB
but not directly around it as in Arabidopsis. To examine the
impact of diRNA production on DSB repair in human cells, we
investigated the effect of Dicer or Ago2 depletion on DSB repair
efficiency. Whereas DR-GFP/U2OS cells treated with control
siRNAs displayed efficient repair resulting in robust production
of GFP-positive cells after I-SceI expression, a significant reduc-
tion in HR of DNA DSB repair was observed after Dicer or Ago2
depletion (Figures 6B and 6C). We tested the protein expression
levels of several DSB repair proteins and found that the levels
–12, March 30, 2012 ª2012 Elsevier Inc. 7
Figure 6. diRNA Production and Regulation of DSB Repair in Human Cells(A) Deep sequencing analysis of diRNAs generated from the DR-GFP reporter construct. The y axis represents the number of normalized small RNA reads per 10
million sequences, numbers in (+) and (�) values represent the reads of small RNAs derived from sense and antisense strands, respectively. Small RNA reads
from RNA isolated from DR-GFP/U2OS cells at 12, 16, 20, or 24 hr following I-SceI transfection or at 20 hr following transfection with control vector (Control) are
shown. The nucleotide positions (bp) of the components in the DR-GFP construct are shown.
(B and C) Relative repair rate in DR-GFP/U2OS cells after treatment with the indicated siRNAs. Forty-eight hours after siRNA treatment, DR-GFP/U2OS
cells were transfected with I-SceI plasmid for 48 hr and processed for flow cytometric analysis of GFP. The repair efficiency was scored as the percentage of
GFP-positive cells in control, Dicer, or Ago2 siRNA-treated cells. Graph represents the mean of three independent experiments. Error bars indicate SEM.
(D) DR-GFP/U2OS cells were treated with Control, Dicer, or Ago2 siRNAs for 48 hr and analyzed by immunoblotting with the indicated antibodies.
See also Figure S7.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
were comparable in control and Ago2 or Dicer siRNA-treated
cells (Figure 6D). Taken together, the identification of diRNAs
in both plants and human cells points to a conserved role for
the small RNA pathway in DSB repair.
8 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
DISCUSSION
Damaged DNA is repaired through coordinated activation of
cell cycle checkpoints and DNA repair machineries, which
Figure 7. A Model for diRNA-Mediated DSB Repair in Arabidopsis
This model is proposed on the basis of genetically identified components
required for diRNA-mediated DSB repair and their roles extrapolated from their
known functions in the RdDM pathway in Arabidopsis. Single-stranded RNA
transcripts (ssRNAs) are presumably generated by RNA polymerase IV
(NRPD1 and NRPD2) from the sequences in the vicinity of a DSB. Redundant
activities of RNA-dependent RNA polymerases (RDRs) convert the ssRNAs
into double-stranded RNAs (dsRNAs), which are processed into diRNAs by
coordinated actions of Dicer-like proteins (DCL2, DCL3, and DCL4). diRNAs
are then incorporated into Argonaute 2 (AGO2). AGO2/diRNA complexes are
localized to the DSB site through interaction with scaffold transcripts that are
made by Pol V (NRPE1 and NRPE2). AGO2/diRNA complexes may recruit
chromatinmodifying complexes tomodify local chromatin (A) or directly recruit
DSB repair proteins to the DSB site (B) to facilitate DSB repair. Further
experiments are required to test thismodel. The gray dots represent chromatin
modifications.
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
involve protein sensors, transducers, and effectors (Ciccia and
Elledge, 2010; Huen and Chen, 2008; Polo and Jackson, 2011).
In this study, we established an important role for small RNAs
in DSB repair, adding an unsuspected RNA component to the
DSB repair signaling pathway. Importantly, we demonstrated
that this layer of DSB repair regulation is conserved: diRNAs
are produced in both plant and human cells and interfering
with their production has severe effects on DSB repair. It is
also noteworthy that in the filamentous fungus Neurospora
crassa, QDE-2-interacting small RNAs (qiRNAs) derived from
rDNA repeats have been detected in cells treated with DNA
damaging agents (Lee et al., 2009). It was proposed that these
small RNAs contribute to DNA damage response by inhibiting
rRNA biogenesis and protein translation (Lee et al., 2009). In
light of our current findings, an alternative interpretation may
be considered. rDNAs are arrayed in tandem in the Neurospora
genome (Galagan et al., 2003), which makes them perfect
targets for HR. Upon exposure to DNA damaging agents,
DSBs may be introduced within the rDNA repeats. This could
then trigger the production of rDNA-specific small RNAs
that mediate DSB repair on damaged repetitive rDNAs. Inter-
estingly, in the fly female germline, mutations in the repeat-
associated siRNA (rasiRNA, an analog of piRNA in mammals)
pathway resulted in elevated g-H2AX levels (Klattenhoff et al.,
2007), raising the possibility that rasiRNAs are involved in
DSB repair.
RDRs andPol IVwere found to be involved in diRNA biogenesis
in Arabidopsis (Figure 3). Based on their roles in making dsRNA
precursors of hc-siRNAs in the RdDM pathway (Law and Jacob-
sen, 2010;Matzke et al., 2009), we speculate that single-stranded
DNA (ssDNA) generated by resection of the DSB might serve as
the template for Pol IV/RDRs-mediated generation of double-
stranded diRNA precursors (Figure 7). In accordance with this
hypothesis, it has previously been shown that the Neurospora
RDR (QDE-1) can produce dsRNA from ssDNA (Lee et al.,
2010). We found that no diRNAs could be detected in atr mutant
plants (Figure 2C). This could suggest that ATR-dependent phos-
phorylation of components of the DSB repair machinery is
required for the recruitment of the diRNA biogenesis machinery
to the DSB sites. Alternatively, ATR-dependent phosphorylation
of components of the diRNA biogenesis machinery itself could
be required for their activityor recruitment tositesofDNAdamage.
diRNAs were specifically generated from the regions close to
the DSB sites (Figures 2 and 6), implying that diRNAs mediate
DSB repair in cis. We showed that DSB repair is not compro-
mised in the ago4 and drm2 mutants (Figures 3E and 3F), sug-
gesting that diRNAs do not function through changing the DNA
methylation at the DSB sites to mediate DSB repair. Accumu-
lating evidence indicates that DSBs trigger a number of histone
modifications around the DSB sites and these modifications
may facilitate DSB repair (Lukas et al., 2011; Polo and Jackson,
2011).We propose that diRNAsmay function as guidemolecules
for these histone modifications at the DSB site, analogous
to hc-siRNAs in the RdDM pathway (Figure 7A). Alternatively,
diRNAs may play a more direct role in recruiting DSB repair
complexes to DSB sites through their effector protein AGO2 (Fig-
ure 7B). The phosphorylation of H2AX around the DSB sites is
one of the earliest events in response to DSBs and facilitates
local recruitment and retention of DSB repair and chromatin
remodeling factors (Fillingham et al., 2006; Paull et al., 2000;
Podhorecka et al., 2010).We found no evidence of compromised
phosphorylation of H2AX in the diRNA-deficient mutants (Fig-
ure 5 and Figure S6), demonstrating that the very early step in
DSB recognition is intact and that the diRNAs most likely affect
events downstream of H2AX phosphorylation.
In summary, we have demonstrated that small RNAs gener-
ated from the sequences flanking a DSB are important for
efficient DSB repair. It will be very exciting to have future studies
dissecting the molecular and biochemical underpinnings of
diRNAs in DSB repair.
EXPERIMENTAL PROCEDURES
Plant Materials, Human Cells, and Growth Conditions
Arabidopsis mutants and lines used in this study have been previously
described. See Table S4 for references and detailed information about these
mutants. All plants were grown in soil or Murashige and Skoog (MS) medium
at 16 hr light/8 hr dark photoperiod.
Human DR-GFP/U2OS cells (U2OS-derivative cell line harboring an inte-
grated HR reporter construct [DR-GFP]) (Pierce et al., 1999) were grown in
Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum.
DSB Repair Reporter Assay
DGU.US-1 line (DSB reporter line) and 2X35S:I-SceI-8 line (DSB-triggering
line) were used for assaying DSB repair efficiency in Arabidopsis (Mannuss
et al., 2010; Orel et al., 2003). To compare the DSB repair efficiency in a mutant
Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc. 9
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
background with that in the corresponding wild-type background, we first
crossed both the DGU.US reporter (R) line and DSB-triggering (T) line express-
ing I-SceI with the mutant of interest (m/m) independently. After F2 segrega-
tion, the following progenies were identified by genotyping: those homozygous
for either the recombination substrate locus from the R line or the I-SceI-
expressing locus from the T line in the respective homozygous mutant
background (named R/R; m/m and T/T; m/m, respectively) and those in the
WT background (named R/R; M/M and T/T; M/M, respectively). Then, plants
R/R;m/mwere crossedwith T/T;m/m, whereas the correspondingWT siblings
R/R; M/M were crossed with T/T; M/M. The seeds of these crosses were
harvested, sterilized, and sown on MS medium. Thirteen-day-old seedlings
were collected for GUS staining or PCR analysis. For GUS staining, seedlings
were infiltrated with 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, and
0.5 mg/ml X-gluc (Apollo Scientific), followed by incubation at 37�C in the
dark overnight. Then plantlets were cleared in ethanol and the blue sectors
in each plantlet were counted under a stereomicroscope (Nikon) and represen-
tative pictures were taken. To determine the DSB repair rate at the DNA level,
200 ng of genomic DNA were digested with recombinant I-SceI (NEB) at 37�Covernight to remove DNAs that were not repaired. Repaired DNAs were
amplified using p1 and p2 primers as depicted in Figure 1A. The sequences
of the primers are listed in Table S5.
DR-GFP/U2OS cells (Pierce et al., 1999) were used for assaying DSB repair
efficiency in humans. DR-GFP/U2OS cells were transfected with pCBASce
plasmid expressing I-SceI for 48 hr and processed for flow cytometric analysis
of GFP. The repair efficiency was scored as the percentage of GFP-positive
cells. To examine the role of Dicer and Ago2 in DSB repair, DR-GFP/U2OS
cells were treated with siRNAs targeting Dicer and Ago2 prior to the transfec-
tion with pCBASce. The sequences of the siRNAs are listed in Table S5.
All siRNAs duplexes were used at a final concentration of 30 nM and trans-
fections were performed using Lipofectamine RNAiMAX (Invitrogen) according
to the manufacturer’s instructions.
RNA Analyses
Arabidopsis total RNA was extracted from 13-day-old seedlings with Trizol
reagent (Invitrogen). Northern blot analysis with enriched small RNAs or
RNAs extracted from the purified AGO2 complex was carried out as described
(Qi et al., 2005). To detect small RNAs generated from the DSB region, we
amplified a 444 bp PCR fragment from the DGU.US construct, randomly
labeled with 32P-a-dCTP, and used as probe. miR173, SIMPLEHAT2, and
ta-siR255 were probed with end-labeled oligonucleotides. The sequences of
the primers and probes are listed in Table S5. For qRT-PCR, total RNA was
treated with RNase-free DNase I (Promega) and reverse-transcribed by
M-MLV Reverse Transcriptase (Promega) with oligo (dT). qRT-PCR was
performed with SYBR Premix EX Taq (TAKARA) on Applied Biosystems
7500 Fast. The GAPDH gene was detected in parallel and used as the internal
control. The sequences of the primers are listed in Table S5.
Total RNA from human DR-GFP/U2OS cells that were transfected with
pCBASce plasmid was extracted using Tri-reagent (Sigma) at 12, 16, 20,
and 24 hr posttransfection. Cells transfected with a plasmid having the same
vector backbone but missing I-SceI were collected at 20 hr posttransfection
and used as a negative control.
Immunopurification of the AGO2 Complex and Associated Small
RNAs
Immunopurification of the AGO2 complex was performed as previously
described (Mi et al., 2008). A small fraction of the immunoprecipitates was
separated on an 8%SDS-PAGE gel and examined by silver staining for quality
control. The associated small RNAs were extracted from the purified AGO2
complex by Trizol reagent (Invitrogen), resolved on a 15% denaturing PAGE
gel, and visualized by SYBR-Gold (Invitrogen) staining. Gel slices within the
range of 18–28 nt were excised, and the RNAs were eluted and purified for
small RNA library construction.
Small RNA Cloning and Deep Sequencing
Cloning of small RNAs from Arabidopsis and human cells was carried out
essentially as described (Mi et al., 2008). The Illumina GA IIx was used for
sequencing. A detailed protocol is available upon request.
10 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
Bioinformatic Analysis of Small RNAs
All reads obtained from Illumina GA IIx were sorted into respective libraries
by parsing their barcodes. Then the adaptor sequences were removed
using ‘‘vectorstrip’’ in the EMBOSS package. Small RNA reads with length
of 18–28nt were mapped to the Arabidopsis or human nuclear genome.
Unaligned reads were mapped to the DGU.US, DU.GUS, or DR-GFP
sequence. Small RNA densities along the DGU.US, DU.GUS, or DR-GFP
sequence were calculated and plotted within the 100 bp sliding windows
with a step size of 1 bp.
Protein Immunoblots
Thirteen-day-old seedlings of pAGO2::3HA-AGO2 Arabidopsis transgenic line
expressing HA-tagged AGO2 under its native promoter (Montgomery et al.,
2008) were treated with 100 Gy g-irradiation and placed in a growth chamber
at 22�C for 1.5 hr. Seedlings without g-irradiation treatment were used as
controls. Total proteins were extracted with 2X SDS-PAGE loading buffer
(100 mM Tris-HCl [pH 6.8], 100 mM b-mercaptoethanol, 4% [w/v] SDS,
0.2% [w/v] bromophenol blue, and 20% [v/v] glycerol). Proteins in human cells
were extracted in RIPA buffer (20 mM Tris-HCl [pH7.4], 20% glycerol, 0.5%
NP40, 1 mM MgCl2, 0.5 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and
1 mM PMSF). Protein samples were separated by SDS-PAGE, transferred to
PVDF membrane, incubated with antibodies in TBST containing 5% nonfat
dried milk, and, after incubation with HRP-conjugated secondary antibodies,
detected by ECL Western Blotting Detection Kit (BD Bioscience). Antibodies
used in this study included: rabbit anti-Ku80 antibody (CTS 2753), mouse
anti-RPA32 antibody (Abcam, ab2175), mouse anti-Rad50 antibody (Upstate,
05-525), mouse anti-Ago2 antibody (Abcam, ab57113), mouse anti-Dicer anti-
body (Abcam, ab14601), mouse anti-b-tubulin antibody (Sigma T5293 and
T5168), and mouse anti-HA antibody (Roche, 11666606001).
g-H2AX Immunofluorescence Staining and Fluorescence
Microscopy
Arabidopsis leaf nuclei were isolated essentially as previously described
(Onodera et al., 2005). g-H2AX immunofluorescence staining with isolated
leaf nuclei or mitotic root tip nuclei was performed as previously described
(Amiard et al., 2010; Friesner et al., 2005).
Images of the nuclei were acquired with a Nikon Eclipse 90i epifluorescence
microscope equipped with a Nikon DS-Qi1Mc monochrome quantitative
digital camera and filters for Alexa 568, exciter, 545/30 nm/nm; emitter,
610/75 nm/nm and for DAPI, exciter, 360/40 nm/nm; emitter, 450/60 nm/nm.
ACCESSION NUMBERS
Data sets of small RNAs generated in this study are deposited in the National
Center for Biotechnology Infromation Gene Expression Omnibus (http://www.
ncbi.nlm.nih.gov/geo/) under accession number GSE 36338.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and five tables and can be found with this article online at
doi:10.1016/j.cell.2012.03.002.
ACKNOWLEDGMENTS
We thank Drs. A. Britt, L. Du, M. Carmell, and B. Ding for critical reading of the
manuscript and stimulating discussions. We are grateful to Dr. H. Puchta for
DGU.US, DU.GUS and I-SceI trigger lines and Dr. M. Jasin for DR-GFP/
U2OS cell line and PCBASce plasmid. This work was supported by National
Basic Research Program of China (973 program 2012CB910900,
2011CB100700, and 2011CB812600 to Y.Q. and 2011CB510103 to Y.G.Y.).
Received: November 3, 2011
Revised: January 15, 2012
Accepted: March 7, 2012
Published online: March 22, 2012
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
REFERENCES
Amiard, S., Charbonnel, C., Allain, E., Depeiges, A., White, C.I., and Gallego,
M.E. (2010). Distinct roles of the ATR kinase and the Mre11-Rad50-Nbs1
complex in the maintenance of chromosomal stability in Arabidopsis. Plant
Cell 22, 3020–3033.
Baulcombe, D. (2004). RNA silencing in plants. Nature 431, 356–363.
Cao, X., and Jacobsen, S.E. (2002). Role of the arabidopsis DRMmethyltrans-
ferases in de novo DNA methylation and gene silencing. Curr. Biol. 12, 1138–
1144.
Carthew, R.W., and Sontheimer, E.J. (2009). Origins and Mechanisms of
miRNAs and siRNAs. Cell 136, 642–655.
Ciccia, A., and Elledge, S.J. (2010). The DNA damage response: making it safe
to play with knives. Mol. Cell 40, 179–204.
Culligan, K.M., and Britt, A.B. (2008). Both ATM and ATR promote the efficient
and accurate processing of programmed meiotic double-strand breaks. Plant
J. 55, 629–638.
Culligan, K., Tissier, A., and Britt, A. (2004). ATR regulates a G2-phase cell-
cycle checkpoint in Arabidopsis thaliana. Plant Cell 16, 1091–1104.
Culligan, K.M., Robertson, C.E., Foreman, J., Doerner, P., and Britt, A.B.
(2006). ATR and ATM play both distinct and additive roles in response to
ionizing radiation. Plant J. 48, 947–961.
Fillingham, J., Keogh, M.C., and Krogan, N.J. (2006). GammaH2AX and its role
in DNA double-strand break repair. Biochem. Cell Biol. 84, 568–577.
Friesner, J.D., Liu, B., Culligan, K., and Britt, A.B. (2005). Ionizing radiation-
dependent gamma-H2AX focus formation requires ataxia telangiectasia
mutated and ataxia telangiectasia mutated and Rad3-related. Mol. Biol. Cell
16, 2566–2576.
Galagan, J.E., Calvo, S.E., Borkovich, K.A., Selker, E.U., Read, N.D., Jaffe, D.,
FitzHugh, W., Ma, L.J., Smirnov, S., Purcell, S., et al. (2003). The genome
sequence of the filamentous fungus Neurospora crassa. Nature 422, 859–868.
Hartlerode, A.J., and Scully, R. (2009). Mechanisms of double-strand break
repair in somatic mammalian cells. Biochem. J. 423, 157–168.
Herr, A.J., Jensen, M.B., Dalmay, T., and Baulcombe, D.C. (2005). RNA poly-
merase IV directs silencing of endogenous DNA. Science 308, 118–120.
Huen, M.S., and Chen, J. (2008). The DNA damage response pathways: at the
crossroad of protein modifications. Cell Res. 18, 8–16.
Jazayeri, A., Falck, J., Lukas, C., Bartek, J., Smith, G.C., Lukas, J., and Jack-
son, S.P. (2006). ATM- and cell cycle-dependent regulation of ATR in response
to DNA double-strand breaks. Nat. Cell Biol. 8, 37–45.
Kanno, T., Huettel, B., Mette, M.F., Aufsatz, W., Jaligot, E., Daxinger, L., Kreil,
D.P., Matzke, M., and Matzke, A.J. (2005). Atypical RNA polymerase subunits
required for RNA-directed DNA methylation. Nat. Genet. 37, 761–765.
Kapranov, P., Cheng, J., Dike, S., Nix, D.A., Duttagupta, R., Willingham, A.T.,
Stadler, P.F., Hertel, J., Hackermuller, J., Hofacker, I.L., et al. (2007). RNA
maps reveal new RNA classes and a possible function for pervasive transcrip-
tion. Science 316, 1484–1488.
Kapranov, P., Ozsolak, F., Kim, S.W., Foissac, S., Lipson, D., Hart, C., Roels,
S., Borel, C., Antonarakis, S.E., Monaghan, A.P., et al. (2010). New class of
gene-termini-associated human RNAs suggests a novel RNA copying mecha-
nism. Nature 466, 642–646.
Kinner, A., Wu, W., Staudt, C., and Iliakis, G. (2008). Gamma-H2AX in recog-
nition and signaling of DNA double-strand breaks in the context of chromatin.
Nucleic Acids Res. 36, 5678–5694.
Klattenhoff, C., Bratu, D.P., McGinnis-Schultz, N., Koppetsch, B.S., Cook,
H.A., and Theurkauf, W.E. (2007). Drosophila rasiRNA pathway mutations
disrupt embryonic axis specification through activation of an ATR/Chk2 DNA
damage response. Dev. Cell 12, 45–55.
Law, J.A., and Jacobsen, S.E. (2010). Establishing, maintaining and modifying
DNAmethylation patterns in plants and animals. Nat. Rev. Genet. 11, 204–220.
Lee, H.C., Chang, S.S., Choudhary, S., Aalto, A.P., Maiti, M., Bamford, D.H.,
and Liu, Y. (2009). qiRNA is a new type of small interfering RNA induced by
DNA damage. Nature 459, 274–277.
Lee, H.C., Aalto, A.P., Yang, Q., Chang, S.S., Huang, G., Fisher, D., Cha, J.,
Poranen, M.M., Bamford, D.H., and Liu, Y. (2010). The DNA/RNA-dependent
RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in
a process requiring replication protein A and a DNA helicase. PLoS Biol. 8,
e1000496.
Lieber, M.R. (2010). The mechanism of double-strand DNA break repair by the
nonhomologous DNA end-joining pathway. Annu. Rev. Biochem. 79, 181–211.
Lukas, J., Lukas, C., and Bartek, J. (2011). More than just a focus: The chro-
matin response to DNA damage and its role in genome integrity maintenance.
Nat. Cell Biol. 13, 1161–1169.
Ma, J.B., Ye, K., and Patel, D.J. (2004). Structural basis for overhang-specific
small interfering RNA recognition by the PAZ domain. Nature 429, 318–322.
Ma, J.B., Yuan, Y.R., Meister, G., Pei, Y., Tuschl, T., and Patel, D.J. (2005).
Structural basis for 50-end-specific recognition of guide RNA by the A. fulgidus
Piwi protein. Nature 434, 666–670.
Malone, C.D., and Hannon, G.J. (2009). Small RNAs as guardians of the
genome. Cell 136, 656–668.
Mannuss, A., Dukowic-Schulze, S., Suer, S., Hartung, F., Pacher, M., and
Puchta, H. (2010). RAD5A, RECQ4A, and MUS81 have specific functions in
homologous recombination and define different pathways of DNA repair in
Arabidopsis thaliana. Plant Cell 22, 3318–3330.
Matzke, M., Kanno, T., Daxinger, L., Huettel, B., and Matzke, A.J. (2009).
RNA-mediated chromatin-based silencing in plants. Curr. Opin. Cell Biol. 21,
367–376.
Mi, S., Cai, T., Hu, Y., Chen, Y., Hodges, E., Ni, F.,Wu, L., Li, S., Zhou, H., Long,
C., et al. (2008). Sorting of small RNAs into Arabidopsis argonaute complexes
is directed by the 50 terminal nucleotide. Cell 133, 116–127.
Mochizuki, K., and Gorovsky, M.A. (2004). Conjugation-specific small RNAs in
Tetrahymena have predicted properties of scan (scn) RNAs involved in
genome rearrangement. Genes Dev. 18, 2068–2073.
Montgomery, T.A., Howell, M.D., Cuperus, J.T., Li, D., Hansen, J.E.,
Alexander, A.L., Chapman, E.J., Fahlgren, N., Allen, E., and Carrington, J.C.
(2008). Specificity of ARGONAUTE7-miR390 interaction and dual functionality
in TAS3 trans-acting siRNA formation. Cell 133, 128–141.
Moynahan, M.E., and Jasin, M. (2010). Mitotic homologous recombination
maintains genomic stability and suppresses tumorigenesis. Nat. Rev. Mol.
Cell Biol. 11, 196–207.
Onodera, Y., Haag, J.R., Ream, T., Costa Nunes, P., Pontes, O., and Pikaard,
C.S. (2005). Plant nuclear RNA polymerase IV mediates siRNA and DNAmeth-
ylation-dependent heterochromatin formation. Cell 120, 613–622.
Orel, N., Kyryk, A., and Puchta, H. (2003). Different pathways of homologous
recombination are used for the repair of double-strand breaks within tandemly
arranged sequences in the plant genome. Plant J. 35, 604–612.
Paull, T.T., Rogakou, E.P., Yamazaki, V., Kirchgessner, C.U., Gellert, M., and
Bonner, W.M. (2000). A critical role for histone H2AX in recruitment of repair
factors to nuclear foci after DNA damage. Curr. Biol. 10, 886–895.
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H.L., and Poethig, R.S. (2004).
SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the
production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379.
Pierce, A.J., Johnson, R.D., Thompson, L.H., and Jasin, M. (1999). XRCC3
promotes homology-directed repair of DNA damage in mammalian cells.
Genes Dev. 13, 2633–2638.
Podhorecka, M., Skladanowski, A., and Bozko, P. (2010). H2AX Phosphoryla-
tion: Its Role in DNA Damage Response and Cancer Therapy. J. Nucleic Acids
2010.
Polo, S.E., and Jackson, S.P. (2011). Dynamics of DNA damage response
proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25,
409–433.
Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc. 11
Please cite this article in press as: Wei et al., A Role for Small RNAs in DNA Double-Strand Break Repair, Cell (2012), doi:10.1016/j.cell.2012.03.002
Puchta, H. (2005). The repair of double-strand breaks in plants: mechanisms
and consequences for genome evolution. J. Exp. Bot. 56, 1–14.
Qi, Y., Denli, A.M., and Hannon, G.J. (2005). Biochemical specialization within
Arabidopsis RNA silencing pathways. Mol. Cell 19, 421–428.
Qi, Y., He, X., Wang, X.J., Kohany, O., Jurka, J., and Hannon, G.J. (2006).
Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed
DNA methylation. Nature 443, 1008–1012.
Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J., and
Joshua-Tor, L. (2005). Purified Argonaute2 and an siRNA form recombinant
human RISC. Nat. Struct. Mol. Biol. 12, 340–349.
San Filippo, J., Sung, P., and Klein, H. (2008). Mechanism of eukaryotic homol-
ogous recombination. Annu. Rev. Biochem. 77, 229–257.
Sasaki, M., Lange, J., and Keeney, S. (2010). Genome destabilization by
homologous recombination in the germ line. Nat. Rev. Mol. Cell Biol. 11,
182–195.
Song, J.J., Smith, S.K., Hannon, G.J., and Joshua-Tor, L. (2004). Crystal struc-
ture of Argonaute and its implications for RISC slicer activity. Science 305,
1434–1437.
Taft, R.J., Glazov, E.A., Cloonan, N., Simons, C., Stephen, S., Faulkner, G.J.,
Lassmann, T., Forrest, A.R., Grimmond, S.M., Schroder, K., et al. (2009).
12 Cell 149, 1–12, March 30, 2012 ª2012 Elsevier Inc.
Tiny RNAs associated with transcription start sites in animals. Nat. Genet.
41, 572–578.
Tolia, N.H., and Joshua-Tor, L. (2007). Slicer and the argonautes. Nat. Chem.
Biol. 3, 36–43.
Vazquez, F., Vaucheret, H., Rajagopalan, R., Lepers, C., Gasciolli, V., Mallory,
A.C., Hilbert, J.L., Bartel, D.P., and Crete, P. (2004). Endogenous trans-acting
siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79.
Wierzbicki, A.T., Haag, J.R., and Pikaard, C.S. (2008). Noncoding transcription
by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of over-
lapping and adjacent genes. Cell 135, 635–648.
Wu, L., Zhou, H., Zhang, Q., Zhang, J., Ni, F., Liu, C., and Qi, Y. (2010). DNA
methylation mediated by a microRNA pathway. Mol. Cell 38, 465–475.
Xie, Z., Johansen, L.K., Gustafson, A.M., Kasschau, K.D., Lellis, A.D., Zilber-
man, D., Jacobsen, S.E., and Carrington, J.C. (2004). Genetic and functional
diversification of small RNA pathways in plants. PLoS Biol. 2, E104.
Yao, M.C., and Chao, J.L. (2005). RNA-guided DNA deletion in Tetrahymena:
an RNAi-based mechanism for programmed genome rearrangements. Annu.
Rev. Genet. 39, 537–559.
Zilberman, D., Cao, X., and Jacobsen, S.E. (2003). ARGONAUTE4 control of
locus-specific siRNA accumulation and DNA and histonemethylation. Science
299, 716–719.
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