RESEARCH HIGHLIGHT

Cell Research (2014) :1-2.
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diRNA-Ago2-RAD51 complexes at double-strand
break sites
Cell Research advance online publication 11 April 2014; doi:10.1038/cr.2014.45

The way in which the DNA damage
response signals the presence of DNA
lesions and mediates DNA repair has
not been fully elucidated. Now, Gao etal. reveal that diRNA-Ago2 complexes recruit RAD51 to the break sites.
Eukaryotic genomic DNA is boundand protected by a variety of chromatinproteins, but cells in our body receive
constant threats from endogenous andenvironmental agents on their DNA[1-4]. Therefore, cells have evolved
mechanisms, collectively referred toas the DNA damage response (DDR)
that detects DNA lesions, signals theirpresence, and mediates repair. Doublestrand DNA breaks (DSBs) represent
the most harmful DNA lesions because
if not repaired correctly, they can trigger permanent growth arrest and cell
death, and even induce oncogenictranslocations of chromosomes. Thus,
DSB signaling and repair are crucial tomaintain genome integrity.
There are two principal eukaryotic
repair mechanisms: non-homologous
end-joining (NHEJ) which is errorprone and can operate in any phaseof the cell cycle, and homologous recombination (HR) which is restricted
to the late S and G2 phases of growing
cells because it uses sister chromatid
sequence as a template to mediatefaithful repair. The basic biochemical
requirements of DSB repair directedby HR can be broken down into four
steps [1-3]: (1) sensing and resectingof the DSB by nuclease and helicase
complexes to produce long stretchesof 3′ single-stranded DNA (ssDNA).
These ssDNAs are coated by replica-tion protein A (RPA) with concomitant
recruitment of protein kinases suchas ataxia telangiectasia (ATR), which
in turn phosphorylate a histone H2Avariant H2AX to generate γH2AX on
the chromatin in cis, starting from theregion most proximal to the DNA lesion
and spreading distally; (2) RPA is thendisplaced by RAD51 to form a RAD51ssDNA filament; (3) the RAD51-ssDNA
filament then searches for and synapses
with a homologous sequence within
the undamaged double-stranded DNA(dsDNA) template to form a physicalconnection between the invading ssDNA and the homologous dsDNA target, generating a displacement D-loop
in which DNA synthesis is initiated toreplace the DNA surrounding the former
break site; (4) finally, resolution of theD-loop occurs either by dissociation of
one of the invading strands or throughmigration of double Holliday junction
intermediates.
Each of these steps requires theconcerted activity of several factors, including small RNAs. Qi and colleagues
recently discovered a class of 21 nt-longsmall RNAs, which are induced byDSBs and originate from the vicinity ofthe DSBs by a Dicer-dependent mechanism [5]. These small RNAs, termed
DSB-induced small RNAs (diRNAs),
are loaded onto Ago2, the key component of the RNA-induced silencing
complex (RISC) in RNAi [6]. Dicer orAgo2 knockdown reduces DSB repair
efficiency, suggesting a model in which
diRNAs trigger DSB repair by guiding
Ago2 complexes (diRISCs) to target
DSB sites by means of base pairing.

This model also implies that diRISCsrecruit DSB repair factors to the target
DSB sites. Similar small RNAs inducedby DDR have been reported in othersystems [7-9]. However, the specificfunction of small RNAs has remainedlargely unknown.
Gao et al. [10] now take advantageof the DR-GFP/U2OS HR reporter
system to determine whether Ago2 andother RNAi factors contribute to DSB
repair. This system uses a modified
nonfunctional green fluorescent protein(GFP) gene with an I-SceI endonucleaserecognition site to introduce DSBs [11].
When cleaved, an HR repair event witha linked donor GFP fragment restores
functional GFP expression, which isreadily detected by flow cytometry. Theauthors found that depletion of tested
RNAi factors impairs HR repair in thereporter system. It was also revealed
that the function of diRNAs in HR repair specifically relies on Ago2 amongfour Argonautes (Ago1-4) expressedin human cells. Ago2 is the only Argonaute that exhibits small RNA-directedendonuclase activity or Slicer activity in
mammals [5].
To determine whether diRNAs
could directly participate in HR repair,
the authors incubated Dicer-depleted
cells with small RNAs isolated fromI-SceI-transfected reporter cells. Small
RNAs isolated from DSB-introduced
cells could restore HR repair in Dicerdepleted cells. These findings show that
RNAi directly mediates HR repair andsuggest that both diRNAs and Ago2,
and therefore presumably diRISCs, arerequired for certain steps in the repair.

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2Figure 1 Two models of diRNA biogenesis and its Ago2 loading pathways. Bothmodels depend on bi-directional transcription (magenta arrow) around the DSB
site (red scissors) and the presence of a double-stranded intermediate transcript ofdiRNA (pre-diRNA). The main difference between models A and B is the location ofAgo2-diRNA complex formation.

An important step toward understanding how Ago2 mediates HR repair
is to determine its precise localization
on chromatin in DSB-introduced cells.
The authors performed chromatin immunoprecipitation and observed enrichment of Ago2 at I-SceI-induced DSB
sites. This prompted them to examine
whether Ago2 interacts with DSB repair
proteins. In untreated cells, Ago2 interacted specifically with RAD51 among
other tested DDR proteins and thisinteraction was enhanced by ionizing
radiation (IR) treatment that generated
DSBs. RAD51, a eukaryotic homologof E. coli RecA, plays a central role inthe strand exchange during HR and accumulates at DSB sites (RAD51 foci)upon DNA damage [3, 4]. To examine
whether the efficient formation of
RAD51 foci depends on diRNAs and/or
Ago2, the authors performed immunostaining and found that depletion of Ago2
or Dicer severely impaired RAD51
recruitment at DSBs in IR-treated cells.
However, accumulation of other DDR
factors including γH2AX and RPA wasunaffected. Although this contradicts
an earlier report that Dicer and Drosha
regulate the recruitment of many DDR
proteins [8], the current findings suggestthat Ago2 directly recruits RAD51 toDSBs probably with the aid of diRNAs

that originate from the DSBs (Figure 1).
Finally, to determine whether small
RNA binding and/or Slicer activity ofAgo2 is required for the recruitment ofRAD51 to DSB sites, the authors used
Ago2 mutants deficient for small RNA
binding or Slicer. These mutants interacted with RAD51, indicating that bothsmall RNA binding and Slicer activity
are dispensable for the Ago2-RAD51
association. However, the Ago2 mutants
did not restore recruitment of RAD51
to DSB sites nor rescue HR repair inAgo2-depleted cells. Based on thesedata, the authors propose a new modelfor RAD51 recruitment to target DSB
sites: namely, transcripts produced fromthe DSB vicinity form dsRNAs thatare converted into diRNA duplexes byDicer. These duplexes are loaded onto
Ago2 and are unwound by Ago2 Slicer
activity, where one strand is cleaved by
Slicer and displaced by Ago2. diRISCs
with RAD51 then target DSBs usingbase pairing with DNA sequences
surrounding the break sites or RNA
transcripts generated from around thebreak sites.
The work by Gao and colleagues
opens the door to studies of how DSB
responses signal the presence of DNA
lesions and mediate their repair. However, there are many future challenges.

For example, how is the transcriptionof diRNA precursors initiated at DSB
sites? Where are diRNAs produced inthe cell (Figure 1)? It is conceivablethat diRNAs are produced and loaded
onto Ago2 to form diRISCs in the cytoplasm, similar to mammalian miRNAs
which are produced from precursors byDicer. If so, how are diRISCs imported
into the nucleus while miRNA-primed
Ago2-RISCs remain in the cytoplasm?
Once imported into the nucleus, how is
the Ago2-RAD51 interaction enhanced
upon IR treatment? Moreover, how are
diRISC-RAD51 repair intermediates
stabilized and retained at DSB sites?
By analogy with heterochromatin assembly in the fission yeast S. pombe inwhich the RNAi pathway targets bothtranscripts of silenced loci and histone
H3 lysine 9 methylation [12], diRISCs
may also target both transcripts at DSBs
and specific histone modifications suchas γH2AX.

Soichiro Yamanaka1,Haruhiko Siomi1

Department of Molecular Biology, Keio University School of Medicine, Tokyo 160-8582, Japan
Correspondence: Haruhiko Siomi
E-mail: awa403@z2.keio.jp

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