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Activation of the SUMO modification system isrequired for the accumulation of RAD51 at sites ofDNA damage

Hiroki Shima1,2, Hidekazu Suzuki1, Jiying Sun1, Kazuteru Kono1, Lin Shi1, Aiko Kinomura1,Yasunori Horikoshi1, Tsuyoshi Ikura3, Masae Ikura3, Roland Kanaar4, Kazuhiko Igarashi2, Hisato Saitoh5,Hitoshi Kurumizaka6 and Satoshi Tashiro1,*1Department of Cellular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan2Department of Biochemistry, Graduate School of Medicine, Tohoku University and CREST, Japan Science and Technology Agency, Sendai 980-8575, Japan3Laboratory of Chromatin Regulatory Network, Department of Mutagenesis, Radiation Biology Center, Kyoto University, Kyoto 606-8501, Japan4Department of Cell Biology and Genetics, Cancer Genomics Center, Department of Radiation Oncology, Erasmus Medical Center, 3000 CARotterdam, The Netherlands5Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan6Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, Tokyo 162-8480, Japan

*Author for correspondence (ktashiro@hiroshima-u.ac.jp)

Accepted 31 August 2013Journal of Cell Science 126, 5284–5292� 2013. Published by The Company of Biologists Ltddoi: 10.1242/jcs.133744

SummaryGenetic information encoded in chromosomal DNA is challenged by intrinsic and exogenous sources of DNA damage. DNA double-

strand breaks (DSBs) are extremely dangerous DNA lesions. RAD51 plays a central role in homologous DSB repair, by facilitating therecombination of damaged DNA with intact DNA in eukaryotes. RAD51 accumulates at sites containing DNA damage to form nuclearfoci. However, the mechanism of RAD51 accumulation at sites of DNA damage is still unclear. Post-translational modifications of

proteins, such as phosphorylation, acetylation and ubiquitylation play a role in the regulation of protein localization and dynamics.Recently, the covalent binding of small ubiquitin-like modifier (SUMO) proteins to target proteins, termed SUMOylation, at sitescontaining DNA damage has been shown to play a role in the regulation of the DNA-damage response. Here, we show that the

SUMOylation E2 ligase UBC9, and E3 ligases PIAS1 and PIAS4, are required for RAD51 accretion at sites containing DNA damage inhuman cells. Moreover, we identified a SUMO-interacting motif (SIM) in RAD51, which is necessary for accumulation of RAD51 atsites of DNA damage. These findings suggest that the SUMO–SIM system plays an important role in DNA repair, through the regulationof RAD51 dynamics.

Key words: RAD51, SUMO, Recombinational DNA repair

IntroductionDouble-strand breaks (DSBs) in chromosomal DNA, caused by

both exogenous and endogenous factors such as ionizingradiation and stalled replication forks, are a serious hazard tothe survival of cells. Homologous recombination (HR) is an

evolutionally conserved mechanism for DSB repair. RAD51 isone of the essential proteins for HR in eukaryotes. Biochemicalexperiments have shown that RAD51 acts as a recombinase inHR, by forming helical filaments on single-stranded DNA

(Wyman and Kanaar, 2006; Atwell et al., 2012). In S-phasecells, RAD51 forms higher-order nuclear structures, calledRAD51 nuclear foci, that increase in number after the

induction of DNA damage by ionizing irradiation or genotoxicdrugs (Haaf et al., 1995; Tashiro et al., 1996; Tashiro et al.,2000). Moreover, RAD51 becomes concentrated at sites

containing DSBs induced by UVA-laser microirradiation(Tashiro et al., 2000; Bekker-Jensen et al., 2006). Thesefindings indicated that RAD51 accumulates at sites containing

DSBs, where the HR pathway is activated. However, themechanism that controls the dynamics of RAD51 localizationupon DSB formation is unknown.

Post-translational modifications often regulate protein

localization and dynamics. Among such protein modifications,the covalent binding of small ubiquitin-like modifier (SUMO)

proteins to target proteins has been shown to modulate proteinfunctions, by altering protein–protein interactions as well as thesubcellular localization of proteins. Mammalian cells possess

three ubiquitous SUMO isoforms: SUMO-1, SUMO-2 andSUMO-3; of these, the latter two are highly conserved andconsidered to be functionally redundant. SUMOylation is

mediated by a cascade of enzymes consisting of the activatingenzyme (E1), conjugating enzyme (E2) and ligating enzyme

(E3). In mammalian cells, UBC9 is the only SUMOylation E2enzyme, and the E3 ligases are considered to be important forproper activity and target selection (Cubenas-Potts and Matunis,

2013). Several lines of evidence suggest the involvement of theSUMO modification system in DNA repair (Kalocsay et al.,

2009; Galanty et al., 2009; Morris et al., 2009; Nagai et al., 2011;Ulrich, 2012; Jackson and Durocher, 2013). SUMOylation ofPCNA inhibits PCNA-dependent DNA repair of lesions induced

by UV exposure or methylmethane sulfonate treatment inbudding yeast (Hoege et al., 2002). SUMOylated PCNA

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functionally cooperates with Srs2, a helicase that blocks theactivity of the HR pathway by disrupting RAD51 nucleoprotein

filaments, to prevent the unwanted recombination of replicatingchromosomes in yeast (Stelter and Ulrich, 2003). Overexpressionof SUMO-1 downregulates the DNA DSB-induced HR pathwayin Chinese hamster ovary cells and H1080 human fibrosarcoma

cells (Li et al., 2000). SUMOylation of XRCC4, a core factor inDNA repair by non-homologous end joining, is important for cellresistance to ionizing radiation and for V(D)J recombination

(Yurchenko et al., 2006). Moreover, recent studies revealed theaccumulation of SUMO proteins together with UBC9, as well asthe E3 enzymes PIAS1 and PIAS4, at DSB sites (Morris et al.,

2009; Galanty et al., 2009; Pinder et al., 2013).

SUMOylation plays a role in the accumulation of repair factorsat sites containing DNA damage, called DNA-damage-inducedfoci (Polo and Jackson, 2011). The E3 enzymes PIAS1 and PIAS4

are involved in the SUMOylation and formation of DNA-damage-induced foci containing p53 binding protein 1 (53BP1) and theBRCA1 tumor suppressor protein, after induction of DNA damage

by UV microirradiation or genotoxic agents (Galanty et al., 2009;Morris et al., 2009). The SUMOylation-negative mutation ofBLM, a RecQ DNA helicase that is defective in Bloom syndrome

patients, affects the formation of DNA-damage-induced BRCA1and c-H2AX foci (Eladad et al., 2005; Manthei and Keck, 2013).RPA70, a subunit of the replication protein A complex (RPA), is

SUMOylated after the induction of replication stress bycamptothecin treatment, which facilitates the formation of DNA-damage-induced RAD51 foci (Dou et al., 2010). A mutation at theSUMOylation site of the MDC1 checkpoint protein impairs the

production of DNA-damage-induced RPA foci (Luo et al., 2012).After the induction of DNA damage, the ubiquitin ligases HERC2and RNF168 are modified with SUMO-1 in a PIAS4-dependent

manner, and promote ubiquitylation to promote protein assemblyat sites of DNA damage induced by ionizing radiation (Danielsenet al., 2012). RNF4, a mammalian SUMO-targeted ubiquitin E3

ligase, also accumulates at sites containing DNA damage after UVmicroirradiation, where it regulates protein turnover and exchange,and is required for effective DSB repair (Galanty et al., 2012; Yinet al., 2012).

Several lines of evidence suggest a relationship between thefunction of RAD51 and the SUMOylation system (Li et al., 2000).Among the SUMO proteins, SUMO-1 was identified as a protein

that interacts with RAD51 (Shen et al., 1996b). The interaction ofRAD51 with UBC9 suggests the involvement of the SUMOylationsystem in the regulation of RAD51 function (Shen et al., 1996a;

Saitoh et al., 2002). Interestingly, the SUMOylation of BLMregulates its interaction with RAD51 and facilitates the HR-mediated repair of stalled replication forks (Ouyang et al., 2009).

Although these findings suggest the possible role of theSUMOylation system in RAD51 accumulation at sitescontaining DNA damage, the means by which the SUMOylationsystem regulates RAD51 function are still unclear.

In our studies of the regulation of RAD51 dynamics inresponse to DNA damage, we examined the involvement ofthe SUMOylation system in the RAD51 accumulation at

DNA lesions. Here, we show that the accumulation of RAD51at damaged DNA sites induced by laser microirradiation isregulated by the SUMO E2 enzyme UBC9 and the E3 enzymes

PIAS1 and PIAS4. These findings suggest the involvementof the SUMOylation system in the accumulation of RAD51 atsites containing DSBs. Moreover, we identified a RAD51

SUMO-interacting (SIM) domain required for the DNA-damage-induced accumulation of RAD51. The DNA-damage-dependent activation of the SUMOylation system could therefore

function in the regulation of the dynamics of RAD51 localizationthrough the SUMO-SIM interaction.

ResultsUBC9 is required for the accumulation of RAD51 at DNA-damage sites

RAD51 has been reported to interact with UBC9, theSUMOylation E2 enzyme (Fig. 1A; Kovalenko et al., 1996).Notably, we identified endogenous UBC9 as a component of theFLAG-HA-RAD51 interactome, purified from nuclear extracts of

HeLa cells after c-irradiation (Fig. 1B). These results led us toinvestigate whether UBC9 is involved in the regulation of theDNA-damage-induced relocalization of RAD51. Therefore, we

decided to apply the UVA-microirradiation system to examine thedynamics of the localization of EGFP-UBC9. Increased signalintensity of EGFP-UBC9 along the microirradiated lines was

observed within 1 minute after UVA-microirradiation (Fig. 1C).This suggests that UBC9 accumulates at sites containing DSBs, inagreement with a previous report (Galanty et al., 2009).

Because RAD51 accumulates at sites containing DNA damagewithin 10–20 minutes after UVA-microirradiation (Tashiro et al.,

2000), the accumulation of UBC9 at sites of DNA damage occursbefore that of RAD51 (Fig. 1C; supplementary material Fig. S1).Therefore, we next investigated the requirement of UBC9 for the

accumulation of RAD51 at DNA-damage sites. For this, we usedshort-interfering RNAs (siRNAs) to deplete UBC9 in the humanfibroblast cell line GM0637 and the human osteosarcoma cell

line U2OS (Fig. 1D,E; supplementary material Fig. S2A,D).Immunofluorescence staining using an anti-RAD51 antibodyrevealed that depletion of UBC9 significantly reduced the

percentage of cells with increased RAD51 signal intensity at themicroirradiated sites (Fig. 1F,G; supplementary material Fig.S2B,E,F). HR is active and RAD51 forms nuclear foci in the S/G2 phase, and depletion of UBC9 does not decrease the percentage

of cells in S/G2 phase (Hayashi et al., 2002; Riquelme et al., 2006;Tashiro et al., 1996; Durant and Nickoloff, 2005). Therefore, ourresults suggest the involvement of UBC9 in the accumulation of

RAD51 at sites containing DNA damage. To confirm thispossibility, we examined the RAD51 focus formation in UBC9-depleted U2OS cells after treatment with ionizing irradiation, and

found that RAD51 focus formation was significantly disturbed bydepletion of UBC9 (supplementary material Fig. S2C). Theseresults suggest that UBC9 is required for RAD51 accumulation at

sites containing DNA damage.

SUMO-1 is required for RAD51 accumulation at sitescontaining DNA damage

RAD51 interacts preferentially with SUMO-1, as compared withSUMO-2 (supplementary material Fig. S3) (Shen et al., 1996b).

Importantly, SUMO-1 accumulated at sites containing DNAdamage induced by UVA microirradiation (supplementarymaterial Fig. S1). Therefore, to elucidate the role of SUMO

modifications in the regulation of RAD51 function, we examinedRAD51 accretion at sites containing DSBs in SUMO-1-depletedcells after UVA microirradiation (Fig. 2A–C). The percentage of

cells with endogenous RAD51 accumulation at UVA-microirradiated sites was significantly lower in SUMO-1-depleted cells compared with the control (Fig. 2B,C). The

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involvement of SUMO-1 in the accumulation of RAD51 at

damaged DNA sites was also supported by the finding that

SUMO-1 depletion significantly reduced the percentage of cells

with RAD51 foci after c-irradiation (Fig. 2D). These findings

indicate that SUMO-1 is required for RAD51 accumulation at

sites with DNA damage.

Requirement of PIAS1 and PIAS4 for RAD51 accumulation

at DNA-damage sites

The SUMOylation E3 ligases PIAS1 and PIAS4 function in the

DNA-damage response (Galanty et al., 2009). Therefore, we next

examined whether PIAS1 or PIAS4 are involved in the

accumulation of RAD51 at DNA-damage sites. For this

purpose, we examined the effect of depletion of PIAS1 or

PIAS4 by siRNA on the accumulation of RAD51 at

microirradiated sites (Fig. 3). Immunofluorescence staining of

UVA-microirradiated cells revealed that the accretion of RAD51

at the microirradiated sites was significantly impaired by the

depletion of PIAS4 (Fig. 3C,D). In PIAS1-depleted cells, the

accumulation of RAD51 at microirradiated sites was also

decreased, but not as much as in PIAS4-depleted cells

(Fig. 3D). Because the percentage of cells in S/G2 is not

decreased by the depletion of PIAS1 or PIAS4 (Galanty et al.,

2009), these findings suggest that PIAS1 and PIAS4 are

Fig. 1. UBC9 is required for the DNA-damage-dependent accumulation of RAD51. (A) Co-immunoprecipitation of Myc-UBC9 and RAD51. Anti-Myc

immunoprecipitation was performed using GM0637 cells transiently expressing Myc-UBC9 and/or FLAG-HA-RAD51, and then co-precipitating RAD51 was

detected by western blotting with anti-RAD51. (B) The RAD51 complex was immuno-affinity purified from the nuclear soluble fraction of HeLa S3 cells stably

expressing FLAG-HA-RAD51, at 30 minutes after ionizing irradiation (12 Gy) treatment. The immunoblotting analysis was performed using an anti-UBC9

antibody. (C) Quantitative analysis of EGFP signal intensity after UVA microirradiation in GM0637 cells, expressing either EGFP-UBC9 (left) or EGFP-SUMO-1

(right). The signal intensity of EGFP in the microirradiated areas was measured. To confirm the DSB-dependent accumulation of UBC9 at microirradiated sites,

the cells were pre-incubated in medium containing Hoechst 33342 (+Hoechst) or not (2Hoechst) before UVA-microirradiation. Results are means 6 s.e. of

three independent experiments. (D) Depletion of UBC9 by siRNA against UBC9. Immunofluorescence staining of GM0637 cells transfected with the UBC9-

specific siRNA [siUBC9 (#1)] was performed using an anti-UBC9 antibody. UBC9 and DNA are shown in red and blue, respectively, in the merged images. Scale

bars: 20 mm. (E) Immunoblotting analysis of GM0637 cells transfected with siUBC9 (#1), using an anti-UBC9 antibody. (F) Immunofluorescence staining of

UBC9-depleted GM0637 cells, at 25 minutes after UVA microirradiation, was performed using anti-RAD51 and anti-cH2AX antibodies. RAD51, cH2AX

and DNA (Hoechst 33342) are shown in green, red and blue, respectively, in the merged images. Scale bars: 20 mm. (G) Quantification of RAD51 accumulation at

microirradiated regions in GM0637 cells. The mean of three independent experiments is indicated, with the error bars representing the s.d.

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important for the regulation of RAD51 dynamics in the DNA-damage response. Taken together, our results suggest that

activation of the SUMOylation system at DNA-damage sites isrequired for the localization of RAD51.

RAD51 interacts with SUMO through its SIM domain

Our findings indicate the involvement of SUMOylation in theaccumulation of RAD51 at sites of DNA damage. AlthoughRAD51 interacted with SUMO-1 in a yeast two-hybrid analysis,

RAD51 lacks putative SUMOylation consensus sequences(Fig. 4B) (Shen et al., 1996b). Therefore, the interactionbetween RAD51 and SUMO has been suggested to be non-

covalent (Ouyang et al., 2009). The SUMO interacting motif(SIM; V/I-A-V/I-V/I) has been identified as the domain requiredfor non-covalent binding with SUMO (Song et al., 2004; Shenet al., 2006; Lin et al., 2006). We found this consensus in RAD51

at amino acid positions 261–264 (VAVV) in the C-terminalregion, and determined that it is conserved from yeast to human(Fig. 4A). This led us to study the effect of the SIM in RAD51 on

its interaction with SUMO-1. First, we constructed RAD51mutants in which the putative SIM was mutagenized, byreplacing the large nonpolar amino acids (VAVV) with small

nonpolar ones (AAAS) or substituting V264 with lysine(V264K), respectively, which has been demonstrated to causeSIM dysfunction (Song et al., 2004; Shen et al., 2006; Lin et al.,

2006). To examine the requirement of the SIM of RAD51 for theinteraction with SUMO-1, we performed a yeast two-hybridanalysis. Neither RAD51-AAAS nor RAD51-V264K interacted

with SUMO-1 (Fig. 4B). These findings suggest that the RAD51SIM domain is required for the interaction with SUMO-1.

The RAD51 SIM domain is required for the regulation ofRAD51 dynamics after induction of DNA damage

To address whether the SIM is required for DNA-damage-

dependent accumulation of RAD51, we microirradiated cellsexpressing EGFP-tagged RAD51-WT, RAD51-AAAS orRAD51-V264K. The accumulation of RAD51-WT at sites of

DNA damage was observed in more than 40% of themicroirradiated cells (18 out of 38 microirradiated cells,supplementary material Fig. S4). By contrast, RAD51-AAAS and

RAD51-V264K failed to accumulate at the microirradiated sites(supplementary material Fig. S4). This suggested that SIM isrequired for the accumulation of RAD51 at DNA damage.Although RAD51 is localized to both the nucleus and cytoplasm

(Mladenov et al., 2009), the EGFP-tagged RAD51-AAASand RAD51-V264K proteins showed cytoplasmic-dominantdistributions, compared with RAD51-WT in GM0637 cells

(supplementary material Fig. S4A). Thus, the two mutants mightfail to accumulate at DNA-damage sites because of theirinsufficient amounts in the nucleus, rather than their impaired

interaction with SUMO. To exclude this possibility, we inserted anuclear localization signal (NLS) into the EGFP-tagged RAD51mutants. After confirming their localization was now mostly

nuclear, we performed UVA-microirradiation analysis (Fig. 5). TheEGFP-NLS-tagged RAD51-WT accumulated at microirradiatedsites, but the RAD51-AAAS and RAD51-V264K mutants both

Fig. 2. SUMO-1 is involved in DNA-damage-dependent

accumulation of RAD51. (A) Depletion of SUMO-1 by

pSIREN-shSUMO-1 (arrows). Cells transfected with pSIREN-

shSUMO-1 are DsRed-positive. Endogenous SUMO-1 was

detected by immunofluorescence staining, using an anti-

SUMO-1 antibody. Scale bars: 20 mm. (B) Damage-dependent

accumulation of RAD51 (arrows) was visualized by

immunofluorescence staining, using an anti-RAD51 antibody,

in control and SUMO-1-depleted cells at 30 minutes after

UVA-microirradiation. RAD51, DsRed and DNA (Hoechst

33342) are shown in green, red and blue, respectively, in the

merged images. Scale bars: 20 mm. (C) Percentage of cells

showing RAD51 accumulation at microirradiated regions.

Results are the total of two independent experiments. n599

for vector, n566 for shSUMO-1 (#1) and n578 for shSUMO-

1 (#2), respectively. (D) Percentage of RAD51-foci-positive

cells expressing either shSUMO-1 (#1) or shSUMO-1 (#2) at

2 hours after c-irradiation (2 Gy). The mean of three

independent experiments is indicated, with error bars

representing the s.d.

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failed to do so (Fig. 5). Taken together, these findings support the

proposal that the RAD51 SIM plays an important role in RAD51

accumulation at sites containing DNA damage.

The RAD51 SIM is required for the recombinational repair

of DSBs

Having established that the RAD51 SIM plays a role in the

regulation of RAD51, we asked whether the RAD51 SIM-SUMO

interaction functionally affects DSB repair. To this end, we

examined the effect of the expression of RAD51-WT or RAD51-

V264K on HR levels in U2OS cells, using a DR-GFP assay. In

this assay, a single DSB is introduced into the chromosomally

integrated DR-GFP substrate by the I-SceI endonuclease, and the

repair of the DSB by HR generates cells expressing functional

GFP (Pierce and Jasin, 2005; Sakamoto et al., 2007; Hosoya et al.,

2012). The transfection of siRNA against RAD51 decreased the

percentage of GFP-positive cells by around 50% (supplementary

material Fig. S5). As expected, the exogenous expression of

Fig. 3. PIAS1 and PIAS4, the E3 enzymes of

SUMOylation, are important for DNA-damage-dependent

accumulation of RAD51. (A) Depletion of PIAS1 by siRNA

in GM0637 cells was confirmed by immunoblotting, using an

anti-PIAS1 antibody. (B) Depletion of PIAS4 by siRNA was

confirmed with anti-FLAG immunoblotting analysis of

GM0637 cells expressing FLAG-PIAS4, transfected with

either control or PIAS4 siRNAs. (C) Immunofluorescence

staining of PIAS1- or PIAS4-depleted GM0637 cells at

30 minutes after UVA microirradiation, using anti-RAD51

and anti-cH2AX antibodies. RAD51, cH2AX and DNA

(Hoechst 33342) are shown in green, red and blue,

respectively, in the merged images. Scale bars: 20 mm.

(D) Percentage of cells with RAD51 accumulation at

microirradiated regions in PIAS1- or PIAS4-depleted GM0637

cells. The mean of three independent experiments is indicated,

with error bars representing the s.d.

Fig. 4. RAD51 interacts with SUMO-1 through its SIM. (A) The

SUMO-interacting motif (SIM) is evolutionally conserved in

eukaryotic RAD51 homologues. Amino acid residues matching the

consensus sequences of SIM (V/I-A-V/I-V/I) are shown in red.

(B) Two-hybrid analysis of RAD51 and SUMO-1. The growth of

two independent transformants on SD +His (left) and SD –His

(middle) plates was examined for each combination of vectors

indicated (right).

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RAD51-WT significantly restored the HR activity repressed by

depletion of endogenous RAD51 (supplementary material Fig.

S5). By contrast, the expression of the RAD51-V264K protein in

U2OS-DRGFP cells failed to do so (supplementary material Fig.

S5). To exclude the possibility that the failure to restore HR

activity was due to the low nuclear amounts of the RAD51

mutants, we performed the DR-GFP assay using NLS-tagged

RAD51 WT, V264K and AAAS mutants. As a result, the

restoration of the HR activity by RAD51 WT, but not by the

RAD51 mutants, was confirmed (Fig. 6). This finding supports the

proposal that the SUMO–SIM interaction, involving the RAD51

SIM domain, is required for the regulation of HR activity in cells.

Taken together, our findings suggest that the in situ activation

of the SUMOylation system around DSBs facilitates the

accumulation of RAD51 through its SIM domain for HR activity.

DiscussionWe have shown that SUMO-1, the E2 SUMO conjugating enzyme

UBC9, and the E3 ligases PIAS1 and PIAS4 are involved in the

accumulation of human RAD51 at DSB-containing sites. We also

identified SIM, a SUMO-interacting motif in RAD51, which is

required for RAD51 accretion at damaged DNA sites. These

findings strongly suggest that the SUMO–SIM system, involving

the interaction of RAD51 with free SUMO-1 or SUMOylated

proteins through the SIM, plays an important role in the regulation

of RAD51 dynamics for DNA repair in human cells.

We showed that of the SUMOylation E3 enzymes, PIAS4

contributes more strongly to the accumulation of RAD51 at sites

containing DSBs, compared with PIAS1 (Fig. 3). We also found

that SUMO-1, rather than SUMO-2, interacts with RAD51

(supplementary material Fig. S3). Because SUMOylation with

SUMO-1 is facilitated by PIAS4 upon DNA damage, RAD51

Fig. 5. The SIM domain of RAD51 is required for the

DNA-damage-dependent accumulation of RAD51.

(A) EGFP fluorescence signals in GM0637 cells transfected

with pEGFP-NLS, pEGFP-NLS-RAD51 (WT), pEGFP-NLS-

RAD51-AAAS (AAAS) or pEGFP-NLS-RAD51-V264K

(V264K) expression vectors were examined at 1 hour after

UVA microirradiation. Immunofluorescence staining of cells

with an anti-cH2AX antibody was performed to visualize the

microirradiated sites. EGFP, cH2AX and DNA (Hoechst

33342) are shown in green, red and blue, respectively, in the

merged images. Arrows indicate the accumulation of EGFP-

NLS-RAD51 at the microirradiated sites. Scale bars: 20 mm.

(B) Percentage of cells expressing either pEGFP-NLS (n531),

pEGFP-NLS-RAD51 (n533), pEGFP-NLS-RAD51-AAAS

(N531) or pEGFP-NLS-RAD51-V264K (n537), showing the

increased signal intensity of EGFP at the microirradiated

regions.

Fig. 6. The SIM domain of RAD51 is required for HR. (A) HR activity of

RAD51-depleted cells (siRAD51), expressing NLS (vector), NLS-RAD51-

WT, NLS-RAD51-V2645K or NLS-RAD51-AAAS, was analyzed using the

DR-GFP assay. Columns represent the mean of the proportions of GFP-

positive cells from four independent experiments. Error bars represent s.d.

(B) Expression of NLS-RAD51-WT, NLS-RAD51-V2645K or NLS-RAD51-

AAAS in RAD51-depleted cells (siRAD51) was confirmed by an immunoblot

analysis using anti-RAD51 antibodies.

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accumulation could be mainly dependent on the PIAS4-mediatedmodification of proteins at sites of DNA damage with SUMO-1

(Galanty et al., 2009). Importantly, both PIAS1 and PIAS4 areinvolved in DNA-damage-dependent SUMOylation (Galanty

et al., 2009). Recently, DNA damage has been shown to triggera SUMOylation wave, leading to simultaneous multisitemodifications of several DNA repair proteins of the HR

pathway in yeast (Psakhye and Jentsch, 2012). The SIMdomain of RAD51 is conserved from yeast to human (Fig. 4A),

it is therefore also possible that in human cells, such multipleSUMOylation reactions of various proteins at DNA-damage sites

could play a role in the regulation of HR. The RAD51 functionshould be strictly regulated within a very narrow range, becauseboth the overexpression and depletion of RAD51 lead to

increased genome instability (Richardson et al., 2004). Severallines of evidence have indicated that in S. cerevisiae the

SUMOylation of PCNA increases the interaction with Srs2,leading to disruption of RAD51 nucleoprotein filaments at stalledreplication forks to prevent unnecessary HR activity (Stelter and

Ulrich, 2003). In addition, overexpression of SUMO-1 has beenshown to downregulate HR activity in mammalian cells (Li et al.,

2000). Therefore, SUMOylation at sites containing DNA damagecould play opposite roles in the regulation of HR, by facilitating

the HR reaction by the accumulation of repair proteins, andby repressing inappropriate HR activity, especially aroundreplication forks. If the RAD51 dynamics were regulated by

only one protein interaction, then it would be difficult to executesuch precise control of its association and dissociation from sites

of DNA damage. RAD51 also interacts with HR-related proteins,such as BRCA2, for its dynamic regulation (Yu et al., 2003; Liu

et al., 2010; Thorslund et al., 2010; Holloman, 2011). The fine-tuning of the accumulation of RAD51 at damaged DNA sites, bydirect multiple interactions with various DNA-repair-associated

proteins and/or through the SIM-SUMO system, could facilitatethe strict regulation of the HR activity for the appropriate repair

of DNA damage.

The radiation-induced nuclear focus formation of RAD51 atthe sites containing DNA damage requires SUMOylation

enzymes. Because focus formation involves the accumulationand retention of proteins, the SUMOylation system could also

regulate the retention of RAD51 at DNA-damage sites, for theformation of higher-order nuclear structures. Several lines ofevidence suggest the involvement of SUMOylation in the

formation of higher-order nuclear architectures. The covalentbinding of SUMO to DNA-repair-related proteins, such as

RAD52, TIP60, RAP80 and PCNA, is induced upon DNAdamage (Hoege et al., 2002; Sacher et al., 2006; Yan et al., 2007;

Cheng et al., 2008). The accumulation of UBC9, PIAS1 andPIAS4 could enhance the SUMOylation of DNA-repair proteinsaround DSBs, which would help to recruit and retain RAD51

around DSBs through its SIM, leading to the formation ofdamage-induced RAD51 foci. Three recent papers shed light on

the function of SIMs in the DNA-damage response (Galanty et al.,2012; Yin et al., 2012; Praefcke et al., 2012). RNF4, a ubiquitin

E3 enzyme, contains SIM domains and targets SUMOylatedDNA-repair proteins such as MDC1 and RPA, leading to theappropriate turnover of these proteins through proteasome-

dependent proteolysis. RPA-SUMO-2/3 is required for thedamage-dependent accumulation of RAD51 (Dou et al., 2010).

However, when RNF4 is depleted, RPA-SUMO-2/3 persists atsites of DNA damage and inhibits the subsequent accumulation

of RAD51 (Galanty et al., 2012). Therefore, RPA-SUMO-2/3might not be the direct target of the RAD51 SIM for RAD51

accumulation at damaged sites. Further exploration of the targets

of RAD51 SIM will provide clues to elucidate the detailedmechanisms of RAD51 regulation.

In this study, we identified a SIM domain in the C-terminalregion of RAD51. This region is important for binding of RAD51

to the nuclear matrix (Nagai et al., 2008; Mladenov et al., 2009).The nuclear matrix is a dynamic subnuclear compartment

supporting the spatial organization of DNA metabolism(Anachkova et al., 2005; Zaidi et al., 2007; Courbet et al.,

2008). However, according to the chromosome territoryinterchromatin compartment (CT-IC) model, the nuclear

architecture is highly organized and based on two principal

components: the chromatin compartment and the interchromatincompartment (Cremer and Cremer, 2010). The chromatin

compartment is composed of interconnected chromosometerritories, which in turn are formed from interconnected

megabase-sized chromatin domains (,1 Mbp) and largerchromatin clusters (Dixon et al., 2012; Markaki et al., 2012).

Transcription reportedly occurs on the surfaces of chromatin

domains or the perichromatin region (Markaki et al., 2012).Because some RAD51 foci are located in the interior of the

interchromatin compartment, damaged DNA could be movedfrom the inside of the chromatin compartment to the

interchromatin compartment or perichromatin region for theHR reaction, as in the case of transcription (Albiez et al., 2006).

Although the role of the nuclear matrix in the regulation of DNA

repair is still unclear, binding of RAD51 to the nuclear matrixcould regulate the dynamics of RAD51 in the interchromatin

compartment for the proper HR reaction (Chiolo et al., 2011).

Materials and MethodsCell culture and ionizing irradiation

GM0637, an SV40-transformed human fibroblast cell line, and the humanosteosarcoma U2OS cell line were cultured in Dulbecco’s modified Eagle’smedium, supplemented with antibiotics (penicillin and streptomycin, Sigma-Aldrich) and 10% fetal calf serum (Tashiro et al., 2000). Ionizing irradiation wasperformed using a 137Cs source at the indicated dose.

Antibodies

Rabbit anti-RAD51 (Calbiochem PC130, BioAcademia 70-001), mouse anti-cH2AX (Upstate Biotechnology, 05-636), rabbit anti-UBC9 (Becton Dickinson,610748), rabbit anti-SUMO-1 (ALEXIS, 210-174-R200), rabbit anti-PIAS1 (CellSignaling, 3550), rabbit anti-PIASy (Abgent, AP1249a), mouse anti-Myc (Roche11667149001), FITC-conjugated goat anti-rabbit (Tago Immunological,ALI4408), Cy3-conjugated sheep anti-mouse (Jackson ImmunoResearch, 515-165-062) and Cy5-conjugated goat anti-mouse (Jackson ImmunoResearch 115-176-003) antibodies were used in the experiments.

Plasmids

pEGFP-RAD51 was constructed by inserting the PCR-amplified RAD51 cDNAinto the BglII site of pEGFP-c1 (Clontech). To construct pEGFP-NLS-RAD51,two oligonucleotides (59-CCGGAGCCCCTCCTAAAAAAAAACGGAAAGTC-GGGA-39 and 59-GATCTCCCGACTTTCCGTTTTTTTTTAGGAGGGGCT-39)were annealed, and the resulting DNA encoding an NLS (APPKKKRKVG) wasinserted into pEGFP-RAD51 via the BspEI/BglII sites. For exogenous expressionof RAD51 in the HR assay, NLS-RAD51 was inserted into pcDNA3.1/Myc-His(-)Cvia its NotI/HindIII sites. The SIM mutations were introduced by site-directedmutagenesis. pGAD424-SUMO-1 and pGAD424-SUMO-2 were kindly provided byDr T. Nishida (Nishida et al., 2000). pGBK-RAD51 was constructed by inserting theRAD51 cDNA into the BamHI/PstI sites of pGBK.

Protein interactions

To examine the interaction between Myc-UBC9 and FLAG-HA-RAD51, GM0637cells co-transfected with pcDNA3.1-Myc-UBC9 and pcDNA3.1-FLAG-HA-RAD51 were lysed in 50 mM HEPES-KOH (pH 7.4), 100 mM NaCl, 10 mMEDTA, 0.5% NP-40, 1 mM DTT and 1 mM PMSF. The cleared lysate was mixed

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with the mouse anti-Myc antibody bound to sheep anti-mouse IgG Dynabeads(Invitrogen) at 4 C for 1.5 hours, and then eluted by 16SDS-PAGE sample buffer.The co-precipitated RAD51 was detected by western blotting. Establishment of theFLAG-HA-RAD51 stable expression cell line and purification of FLAG-HA-RAD51 from the nuclear soluble fraction were performed as described previously(Nakatani and Ogryzko, 2003).

The Matchmaker two-hybrid system (Clontech) was used for the yeast two-hybridassay. AH109 cells were transformed with two plasmids, one containing the wild-typeor mutant RAD51 gene fused with GAL4 binding domain, and the other containingSUMO-1 fused with the GAL4 activating domain. Transformants were streaked onSD/2Trp/2Leu and SD/2Trp/2Leu/2His plates, and grown at 30 C for 2 days.

UVA-microirradiation and live cell imaging

UVA microirradiation and live-cell imaging analysis was performed using a ZEISSLSM510 confocal laser-scanning microscope, with a 636 /1.4 plan-apochromatobjective. Cells on round coverslips were transferred to a Chamlide TC live-cellchamber system (Live Cell Instrument), mounted on the microscope stage, andmaintained at 37 C. The objective was operated with a heater, as part of theChamlide TC. For UVA microirradiation, Hoechst 33258 (Sigma) was addedto cultures at 2 mg/ml. After 10 minutes, the DMEM was replaced by Leibovitz’sL-15 (Gibco) containing 10% FBS and 25 mM HEPES (Gibco). UVAmicroirradiation was performed as described previously (Ikura et al., 2007). The364 nm line of the UVA-laser was used to introduce DSBs. Adobe Photoshop wasused for presentation of the images.

Immunofluorescence microscopy

Cells were fixed for 10 minutes with PBS containing 4% paraformaldehyde, andthen permeabilized with PBS containing 1% SDS and 0.5% Triton X-100 for10 minutes. The cells were then incubated with antibodies in PBS containing 1%BSA at 37 C for 30 minutes. Nuclei were stained with Hoechst 33342. AnAxioplan2 microscope, with AxioCam MRm controlled by Axiovision (Zeiss),was used for visualization. To quantify protein accumulation aftermicroirradiation, at least 20 cells were microirradiated in each experiment andsubjected to immunofluorescence microscopy. The number of nuclei showingcolocalization of RAD51 with cH2AX at microirradiated sites was then counted.

RNAi

The pSIREN-DNR-DsRed-Express vector (Clontech) was used for SUMO-1RNAi. The target sequences were 59-CUGGGAAUGGAGGAAGAAG-39 (#1) and59-CACAUCUCAAGAAACUCAA-39 (#2). The experiments were performed 3days after vector transfection. siGENOME SMARTpool (Thermo ScientificDharmacon M-004910-00) and Silencer Select siRNA (Ambion s14589, 59-AAACAGAUCCUAUUAGGAA-39) were purchased and used as siUBC9 (#1)and siUBC9 (#2), respectively. For PIAS1 and PIAS4 RNAi, the ON-TARGETplus SMART pool (Thermo Scientific Dharmacon L-008167-00 and L-006445-00) was used. siGENOME Non-Targeting siRNA Pool #1 (ThermoScientific Dharmacon D-001206-13) was used as the control RNA. Theexperiments were performed 2 days after transfection with 5–10 nM siRNA.

Homologous recombination repair assay

The HR repair assay was performed as previously reported (Kobayashi et al.,2010). RAD51-depleted U2OS-DRGFP cells created using siRNA (Ambions11734, 59-GGUAGAAUCUAGGUAUGC-39) were reconstituted with wild-typeRAD51, RAD51V264K or RAD51AAAS mutants resistant to siRNA, with orwithout an NLS. To measure the HR repair of I-SceI-generated DSBs, 50 mg of theI-SceI expression vector (pCBASce) was introduced to U2OS-DRGFP cells byelectroporation (GenePulser; Bio-Rad). To determine the efficiency of HR repair,the percentage of GFP-positive cells was quantified by a FACSCantoII cell sorter(Becton Dickinson) at three days after electroporation.

Note added in proofDuring the revision of this manuscript, Dr Stefan Jentsch’s labat the Max Planck Institute of Biochemistry reported the

identification of Rad51 SIM in yeast (Bergink et al., 2013).

AcknowledgementsWe thank Drs Maria Jasin, Junya Kobayashi and Kenshi Komatsu forproviding U2OS-DRGFP cells, and T. Nishida for the pGAD424-SUMO-1 and pGAD424-SUMO-2 vectors.

Author contributionsH. Shima, Y.H., T.I., R.K., K.I., H. Saitoh, H.K. and S.T. planned theexperimental design and wrote the manuscript. H. Shima, H. Suzuki,J.S., K.K., L.S., A.K., Y.H, T.I. and M.I. performed experiments.

FundingThis work was supported in part by Grants-in-Aid from the JapaneseSociety for the Promotion of Science (JSPS), and the Ministry ofEducation, Culture, Sports, Science and Technology (MEXT), Japan.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.133744/-/DC1

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