Sirtuin 6 (SIRT6) Rescues the Decline of Homologous Recombination Repair During Replicative Senescence

Sirtuin 6 (SIRT6) rescues the decline of homologous recombination repair during replicative senescence

Zhiyong Mao, Xiao Tian, Michael Van Meter, Zhonghe Ke, Vera Gorbunova1, and Andrei Seluanov1

Department of Biology, University of Rochester, Rochester, NY 14627

Edited by James E. Cleaver, University of California, San Francisco, CA, and approved June 12, 2012 (received for review January 12, 2012) Genomic instability is a hallmark of aging tissues. Genomic in- (10). SIRT6 has recently been identified as a component of the stability may arise from the inefficient or aberrant function of DNA HR pathway. SIRT6 participates in HR at two steps, by acti- double-stranded break (DSB) repair. DSBs are repaired by homolo- vating PARP1 at the early stages of DSB processing (11) and, gous recombination (HR) and nonhomologous DNA end joining later, by stimulating CtIP (12). (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to Aging organisms exhibit a diminished capacity to repair DSBs. deletions or insertions at the repair site. Here, we used normal It was shown that the recruitment of DSB repair proteins to human fibroblasts with a chromosomally integrated HR reporter DNA damage sites is delayed in lymphocytes of aged donors (13) cassette to examine the changes in HR efficiency as cells progress to and that cells from aged individuals contain a higher number of replicative senescence. We show that HR declines sharply with DSBs (14, 15). We showed previously that NHEJ declines up to increasing replicative age, with an up to 38-fold decrease in fourfold and becomes more error-prone in senescent cells (16). efficiency in presenescent cells relative to young cells. This decline Furthermore, the levels of Ku70 and Ku80, two critical NHEJ is not explained by a reduction of the number of cells in S/G2/M stage proteins, decline in senescent cells (17). However, the changes in as presenescent cells are actively dividing. Expression of proteins HR-mediated repair during replicative senescence and aging involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 have not been directly analyzed. (SIRT6) diminished with cellular senescence. Supplementation of Here, we examined the efficiency of HR repair of induced Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in DSBs in normal human fibroblasts at increasing replicative age. combination, did not rescue the senescence-related decline of HR. HR was measured using chromosomally integrated GFP reporter However, overexpression of SIRT6 in “middle-aged” and presenescent cassettes. We show that HR-mediated repair declines sharply MEDICAL SCIENCES cells strongly stimulated HR repair, and this effect was dependent on with increasing replicative age. This decline occurs even in mono-ADP ribosylation activity of poly(ADP-ribose) polymerase “middle-aged” and presenescent cells, before the cells enter (PARP1). These results suggest that in aging cells, the precise HR senescent growth arrest. The levels of several HR factors decline pathway becomes repressed giving way to a more error-prone with replicative age; however, only supplementation with SIRT6 NHEJ pathway. These changes in the processing of DSBs may con- is able to partly restore HR activity. These studies reveal a pre- tribute to age-related genomic instability and a higher incidence of cipitous decline of HR during replicative aging and suggest that cancer with age. SIRT6 activation provides a potential therapeutic stimulation of SIRT6 may be a potential therapeutic strategy to strategy to prevent the decline in genome maintenance. overcome the age-related decline in DSB repair. Result ging is associated with an increased mutation rate (1) and the appearance of genomic rearrangements (2). The accu- Construction of Primary Human Fibroblast Cell Lines for Measuring HR A fi mulation of mutations and rearrangements is a contributing Ef ciency. To examine the effect of replicative senescence on cause of aging and leads to a decline of tissue functionality and DNA DSB repair by HR, we integrated a HR reporter cassette A an increased incidence of tumors. These mutations and genomic (Fig. 1 ) (6, 10, 18) into the genome of HCA2 cells, a primary fi rearrangements arise from aberrant repair of DNA double- foreskin broblast strain, at population doubling (PD)26. The stranded breaks (DSBs). challenge of studying HR in the context of replicative senes- DSBs are dangerous DNA lesions. If left unrepaired or cence is that the reporter construct must be integrated in pri- repaired incorrectly, DSBs result in a massive loss of genetic mary (nonimmortalized cells). A standard protocol of antibiotic information, chromosomal aberrations, or cell death. DSBs are selection, followed by isolation and expansion of individual clones, requires extensive passaging that would drive primary repaired by two major pathways: nonhomologous end joining fi (NHEJ) and homologous recombination (HR) (3). NHEJ cells into replicative senescence. To overcome this dif culty fi and obtain cells carrying the HR reporter construct at the modi es the broken DNA ends and ligates them together with ∼ no requirement for homology, often generating deletions or youngest possible replicative age, we pooled together 250 insertions (4). In contrast, HR uses an undamaged DNA tem- colonies formed after antibiotic (G418) selection and allowed the cells to reach confluence in a 10-cm plate. Because the plate to repair the break, leading to the reconstitution of the number of cells at confluence on a 10-cm plate is ∼2,000,000, original sequence (5). HR repair is responsible for approxi- we estimated the newly established cell line, named as HCA2- mately one quarter of DNA repair events and has much slower HR, to be at approximately PD39 [PD26 + log (2,000,000/250)]. repair kinetics than NHEJ (6). HR repair begins with the 2 We electroporated 0.1 μg of linearized HR reporter cassette into MRE11, NBS1, and Rad50 complex binding to DNA ends and mediating end resection. The RPA protein is then recruited to DNA ends, in a process regulated by CtIP (7). Once the ends fi Author contributions: Z.M., X.T., V.G., and A.S. designed research; Z.M., X.T., M.V.M., and are resected, Rad51 forms nucleoprotein laments and medi- Z.K. performed research; Z.M., X.T., M.V.M., V.G., and A.S. analyzed data; and Z.M., ates strand invasion of the filament into duplex DNA, usually M.V.M., V.G., and A.S. wrote the paper. on the sister chromatid (8). Rad51 is assisted by Rad52 and The authors declare no conflict of interest. other members of the Rad52 family (9). Missing genomic in- This article is a PNAS Direct Submission. formation is copied from the donor chromosome, leading to 1To whom correspondence may be addressed. E-mail: [email protected] or precise reconstitution of the original sequence. Because mitotic [email protected]. recombination preferentially uses the sister chromatid as a tem- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plate for repair, HR takes place in S/G2/M stages of cell cycle 1073/pnas.1200583109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1200583109 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 A B

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Fig. 1. HR declines with replicative senescence. (A) Reporter cassette for detection of HR. The construct contains two nonfunctional copies of the GFP gene. GFP gene is engineered to contain Pem1 intron. The GFP activity of the first copy is eliminated by a 22-nt deletion and insertion of two I-SceI recognition sites in the first exon. The second GFP gene lacks ATG start codon and the second exon. After induction of DNA DSBs by expressing I-SceI, gene conversion events reconstitute functional GFP gene. (B) Growth curve of HCA2-HR cell line. Gray bars show replicative ages at which young (Y), middle-aged (MA), and presenescent (Pre) cells were used for HR analysis. (C and D)Efficiency of HR decreases with replicative age. Western blots for I-SceI expression using antibodies against HA tag are shown (C and D, Upper). In C, cells HCA2-HR cells at all ages were cotransfected with 5 μg of I-SceI vector and 0.1 μg of pDsRed2-N1. In D, cells were transfected with increasing amounts of I-SceI vector (2 μg for PD45, 4 μg for PD53, 6 μg for PD62) to compensate for somewhat lower expression of I-SceI in older cells. Four days after transfection, cells were analyzed by FACS, and the GFP+/DsRed+ ratio was used as the measure of HR efficiency. HR efficiency in middle-aged and presenescent cells was significantly different from young cells. *P < 0.001. (E) Cutting efficiency of I-SceI does not decline in aged cells. Cells transfected with I-SceI vector as described in D were analyzed by real-time PCR using primers that amplify across the I-SceI recognition site. I-SceI transfection reduces the number of PCR templates. Cutting efficiency was calculated from a ratio between intact reporter cassettes in control and I-SceI– transfected cells. All experiments were repeated at least three times (nine times, three repeats for each pool of integrants) (C and D), and error bars show SD.

HCA2 cells. Our previous analysis of integrated clones indicated were much higher in older cells, and HR efficiency showed that transfection with 0.1 μg of plasmid results in the integration a similar reduction with age (Fig. 1D). To rule out the possibility of a single copy of the reporter per genome (18). Three separate that the cutting efficiency of I-SceI was reduced in older cells transfections were performed, giving rise to three independent because of potential changes in chromatin structure, we mea- pools of integrants. HCA2-HR cell lines were serially passaged sured the reduction in the number of intact I-SceI sites following until they reached senescence at PD71, consistent with the PD at I-SceI transfection using quantitative real-time PCR. The pri- which its parental cell line, HCA2, becomes senescent, indicating mers were designed flanking the I-SceI sites, so that the loci that that the calculation of the starting PD for HCA2-HR lines was were cut by I-SceI would not yield a PCR product. Upon correct. The same protocol was followed for construction of transfection with I-SceI as described in Fig. 1D, genomic DNA HRF and HRIF cell lines. was extracted and the levels of intact I-SceI sites were analyzed (Fig. 1E). The percentage of I-SceI–digested reporter cassettes HR Decreases Sharply with Replicative Age. We then measured the was higher in older cells, indicating that reduced HR efficiency efficiency of HR mediated repair in HCA2-HR cells at PD42–45 was not attributable to inefficient induction of DSBs. These (young), PD52–53 (middle-aged), and PD60–62 (presenescent) results suggest that the decline of HR efficiency is caused by (Fig. 1B). Importantly, the presenescent cells are still in the intrinsic changes associated with replicative aging, and not by logarithmic growth phase and, hence, have a similar fraction of inefficient induction of DSBs in older cells. actively dividing cells as the young and middle-aged cells. We did not include terminally senescent PD > 71 cells in this analysis, Decline of HR Efficiency with Replicative Age Is Not Attributable to – because these cells are cell cycle arrested and would not be Decreased Number of Cells in S/G2/M. The repair of DNA DSBs by expected to repair DSBs by the HR pathway. HCA2-HR cells HR is dependent on the cell cycle because HR requires sister μ μ were transfected with 5 g of I-SceI for inducing DSBs and 0.1 g chromatids, which are only available during the S/G2/M phases of pDsRed2-N1 as an internal control for transfection efficiency. of the cell cycle (10, 19). To examine whether the reduced HR fi Cells were incubated for 4 d to allow suf cient time for GFP and efficiency is caused by reduced number of cells in S/G2/M phase, DsRed expression and analyzed by FACS. The ratio of GFP+ to we examined cell cycle distribution of HCA2-HR cells by pro- DsRed+ cells was used as a measure of HR efficiency. HR ef- pidium iodide (PI) staining and flow cytometry. The mean per- fi C ciency decreased dramatically with replicative age (Fig. 1 ). centage of cells in S/G2/M stages was 33 ± 2.2% at PD41, 29 ± HR efficiency in middle-aged cells was reduced 5-fold compared 6.1% at PD51, and 31 ± 3.2% at PD60 (Fig. 2). These fluctua- with young cells, whereas the repair in presenescent cells was tions in S/G2/M content cannot account for the drop in HR ef- reduced 38-fold compared with young cells. Although HR effi- ficiency and suggest that the changes in cell cycle distribution do ciency was normalized by internal control pDsRed-N2, the ex- not explain the observed age-related decline in HR efficiency. pression level of I-SceI was somewhat lower in older cells (Fig. 1C). To compensate for the lower expression of I-SceI, the Levels of HR Proteins Decline with Cellular Senescence. To un- amount of pCMV-I–SceI plasmid was increased in older cells derstand the mechanisms responsible for the senescence-related (Fig. 1D). Under these conditions, the protein levels of I-SceI decline of HR repair, we examined the levels of HR proteins at

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1200583109 Mao et al. Downloaded by guest on September 30, 2021 Surprisingly, when Rad51, Rad51C, Rad52, and NBS1 were coexpressed together, HR was repressed at all PDs compared with control cells that overexpressed a hypoxanthine phosphor- ibosyltransferase 1 (HPRT) gene (Fig. 4B). We then examined the effect of individual proteins on HR. Supplementing Rad51, Rad51C, Rad52, or NBS1 individually showed that Rad52 sup- pressed HR, Rad51C and NBS1 had no effect, and Rad51 stimulated HR by 1.6-fold in middle-aged cells but not in presenescent cells (Fig. 4B). When we excluded Rad52, a combination of Rad51, Rad51C, and NBS1 stimulated HR by about 1.8- fold in middle-aged cells and had no effect on presenescent cells, which was similar to the effect of Rad51 alone. Remarkably, expression of SIRT6 strongly stimulated HR at all PDs tested, including the presenescent cells (Fig. 4C). SIRT6 stimulated HR by 1.4-fold in young cells, 1.7-fold in middle-aged cells, and 3.9-fold in presenescent cells. Thus, SIRT6 was the only factor capable of stimulating HR repair in presenescent cells. To conﬁrm by an independent assay that SIRT6 overexpression rescues the effects of senescence on DSB repair, we examined the levels of spontaneously occurring γH2AX foci in presenescent cells. SIRT6 reduced the number of γH2AX foci (Fig. 4D). SIRT6 expression did not change cell cycle distribution (Fig. S1), ruling out a possibility that SIRT6 stimulates HR in older cells by promoting cell proliferation. Furthermore, SIRT6 overexpression did not alter the levels of HR proteins (Fig. S2), suggesting that the effect of SIRT6 is mediated by protein–protein interactions at the break site.

We then tested which biochemical activity of SIRT6 is re- MEDICAL SCIENCES quired for the stimulation of HR in presenescent cells. To this end, we transfected presenescent HCA2-HR cells with SIRT6 separation of function mutants G60A, R65A, and S56Y. The G60A has deacetylation activity, R65A has mono-ADP ribosylation activity, and S56Y is catalytically inactive (11). Mono-ADP ribosylation was the only activity required for the stimulation of HR in presenescent cells (Fig. 4E). Because SIRT6 can stimulate HR by two pathways, via in- teraction with CtIP or PARP1 (11), we next examined which pathway is responsible for the stimulation of HR in aging cells.

PD45 PD53 PD62 PD45 PD53 PD62

Rad51 100% 76±22% 49±9% Fig. 2. Changes in cell cycle distribution with replicative senescence. HCA2- HR cells were harvested at indicated PDs 2 d postsplitting, stained with PI, Rad51C 100% 83±12% 56±9% and analyzed by Flow cytometry. The experiment was repeated three times, and the numbers next to the peaks show means and SD. Rad52 100% 80±4% 65±12%

different passages (Fig. 3). Rad51 is a central protein in HR, and MRE11 100% 95±13% 100±14% its levels decreased strongly with increasing cellular age. Fur- thermore, Rad51C, Rad52, and SIRT6 were reduced in older Rad50 100% 92±10% 85±26% cells. The levels of MRE11 and Rad50 remained unchanged, whereas NBS1, which is involved in end resection, was reduced NBS1 100% 85±9% 56±15% in senescing cells. CtIP also declined with replicative senescence. In contrast, the levels of PARP1 protein, which is involved in the CtIP 100% 69±14% 57±10% early response to DNA damage, increased with replicative age, 100% 102±24% 117±40% which suggests that the reduction in the levels of HR proteins PARP1 contributes to the decline of HR repair efficiency. SIRT6 100% 77±9% 61±11% Overexpression of SIRT6 Rescues HR Repair in Aging Cells. To test whether HR efficiency in aging cells can be rescued by supple- Actin 100% 100% 98% menting DNA repair factors, we transfected HCA2-HR cells with expression vectors encoding the HR factors that exhibited Fig. 3. Senescence-related changes in the levels of proteins involved in HR. Cell lysates were prepared from HCA2-HR cells 2 d postsplitting. In each lane, a decline with replicative aging. The ORFs for Rad51, Rad51C, 50 μg of protein was loaded for Western analysis. Actin was used as a load- Rad52, NBS1, SIRT6, and CtIP (a kind gift from Junjie Chen, ing control. Quantification of the changes in middle-aged and presenescent MD Anderson Cancer Center, Houston, TX) were cloned under cells relative to the young cells is shown on the right. Each blot was repeated a CMV promoter and expressed in HCA2-HR cells (Fig. 4A). three times using independent batches of cells, and SDs are shown.

Mao et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Overexpression of CtIP had no effect on HR (Fig. 4C). We were unable to assay the effect of PARP1 overexpression on HR because it caused massive cell death. Instead, we used the speciﬁc PARP1 inhibitor PJ34. Importantly, PJ34 had no inhibitory effect on either deacetylation or mono-ADP ribosylation activities of SIRT6 (Fig. 4 F and G). In the presence of PJ34, SIRT6 overexpression had no effect on HR (Fig. 4C), indicating that SIRT6-mediated rescue of HR in aging cells is dependent on PARP1. To gain an insight into the mechanism by which SIRT6 rescues the decline of HR in presenescent cells, we examined the formation of RAD51 foci after γ-irradiation. RAD51 is the key protein in HR and is often a rate-limiting factor. Interestingly, presenescent cells exhibited a drastic decline in the ability to recruit RAD51 to DNA damage sites (Fig. 4H). This decline was fully rescued by SIRT6 overexpression, indicating that SIRT6 promotes recruitment of DNA repair proteins to DNA lesions (Fig. 4H).

SIRT6 Preferentially Stimulates the Precise HR Pathway. In addition to gene conversion and crossing over, which are precise pathways of HR repair, HR may proceed by a single-stranded annealing (SSA) pathway, leading to deletion of sequences between two direct repeats. The HR reporter we used (Fig. 1A) measured gene conversion, which is the predominant HR repair pathway in mammalian cells (8). To test the effect of SIRT6 overexpression on the more mutagenic SSA pathway, we used two modified reporters: HRF and HRIF (Fig. 5 A and B). HRF detects all three pathways of HR (gene conversion, crossing over, and SSA), whereas HRIF detects gene conversion and crossing over. The constructs were integrated in HCA2 cells as described for HCA2-HR, and the frequency of repair by SSA was measured as HRF minus HRIF. To validate this method of measuring SSA, we tested whether this parameter is stimulated by Rad52, a known mediator of SSA. As expected, Rad52 overexpression stimulated SSA (Fig. 5C). The efficiency of SSA declined with replicative age (Fig. 5D). Interestingly, SIRT6 overexpression did not stimulate SSA (Fig. 5D), indicating that SIRT6 preferentially stimulates the precise pathway of HR repair. In summary, our findings open avenues for preventing age-related genomic instability and decline in DSB repair by activating SIRT6. Discussion We present a systematic analysis of the changes in HR repair during cellular senescence. We used a sensitive fluorescent reporter assay developed by our laboratory (18), which specifically

Fig. 4. SIRT6 rescues the decline of HR efficiency in senescent cells. (A) Western blot showing overexpression of HR-related proteins in HCA2-HR cells. (B) Effect of overexpression of Rad51, Rad51C, Rad52, and NBS1 on HR SIRT6 overexpression reduces the number of γH2AX foci in presenescent at different PDs. Mix is a mixture of Rad51, Rad51C, Rad52, and NBS1 ex- cells. (Left) Quantification. (Right) Representative images. The experiment pression vectors. For expression of individual HR components, 5 μgofthe was repeated three times, and error pars show SD. (E) Mono-ADP ribosyla- expression vectors were cotransfected with 0.1 μg of pDsRed2-N1 and I-SceI tion activity but not deacetylation activity of SIRT6 is required to stimulate expression vector into HCA2-HR cells at different PDs. Older cells were HR in presenescent cells. HCA2 cells were transfected with plasmids encoding transfected with higher amounts of I-SceI expression vector, 2 μg for PD45, SIRT6 separation of function mutants, and the HR assay was performed as 4 μg for PD53, and 6 μg for PD62. For the experiments involving a mixture of described above. The experiment was repeated three times, and error pars HR components, 1.25 μg of each expression vector was cotransfected with show SD. (F) PJ-34 does not inhibit SIRT6 deacetylation activity. To measure DsRed2-N1 and I-SceI expression vectors as described above. Analysis of HR deacetylation activity SIRT6 was overexpressed in HCA2 cells in the presence was performed as described in Fig. 1. (C) SIRT6 rescues HR in senescent cells, of 20 μM PJ34 or 5 mM nicotinamide, and the level of acetylated histone H3 and this effect is mediated by PARP1. HCA2-HR cells were cotransfected with was analyzed by immunoblot. (G) PJ-34 does not inhibit SIRT6 mono-ADP 5 μg of expression vectors encoding SIRT6, PARP1, or CtIP was cotransfected ribosylation activity. Purified SIRT6 was incubated with radiolabeled NAD, into HCA2-HR cells together 0.1 μg of DsRed plasmid and increasing amounts and the level of labeled SIRT6 protein detected by autoradiography. NAM, of I-SceI vector as described in B. PARP1 inhibitor PJ34 was supplemented at nicotinamide, inhibitor of SIRT6 activity. Mono-ADP ribosylation activity of 20 μM starting 1 d before transfection until the cells were harvested for SIRT6 was measured as self-ribosylation reaction in the presence of radio- analysis. All of the experiments were repeated at least three times and error labeled NAD+.(H) SIRT6 overexpression restores the ability of presenescent bars show SD. Asterisk indicates HR efficiencies significantly different from cells to recruit Rad51 to DNA breaks. HCA2 cells at PD62 were irradiated with corresponding controls (P < 0.01); two asterisks indicate HR values signifi- 8Gyofγ-irradiation and hybridized in situ with Rad51 antibodies. The cantly different from cells transfected with SIRT6 vector alone (P < 0.01). (D) experiments were repeated three times, and error bars show SDs.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1200583109 Mao et al. Downloaded by guest on September 30, 2021 I-SceI I-SceI a higher mutation rate, dysregulation of transcription patterns, A fi PCMV PSV40 and, nally, loss of cell and tissue function. Furthermore, the Pem1 Pem1 Neo/Kana Ori shift to the error-prone NHEJ pathway of DSB repair is likely to G SD SA FP G SD SA FP -ATG 22 PE.coli lead to generation of large deletions, joining of inappropriate DNA ends, and chromosomal rearrangements. We hypothesize B I-SceI I-SceI that these changes in DSB processing are a possible mechanism PCMV PSV40 responsible for age-related genomic instability and higher in- Pem1 Pem1 Neo/Kana Ori cidence of cancer in the elderly. G SD SA FP G SD SA FP -ATG 22 PE.coli Studies of DSB processing in the context of organismal aging indicate a decline in repair. Sedelnikova et al. reported that the 0.04 0.12 rate of recruitment of DSB repair proteins to γH2AX foci were C P=0.014 D 0.1 slower in older donors; furthermore, the frequency of sponta- 0.03 0.08 neous γH2AX foci increased with age (13). Earlier studies also 0.02 0.06 observed that cells from older donors contain a higher number of – 0.04 DSBs (27), and analysis of X-ray induced DSBs in lymphocytes 0.01 SSA efficiency SSA SSA efficiency SSA (GFP+/DsRed+) showed that these cells exhibit an age-associated decline in re- (GFP+/DsRed+) 0.02 pair efficiency (14, 28). Thus, our studies provide mechanistic 0 0 Control Rad52 43 51 63 insight into the observed decline in DSB repair, showing that HR Population Doubling repair is sharply reduced in older cells. Fig. 5. SIRT6 does not stimulate SSA pathway. (A) Reporter cassette HRF for Interestingly, the frequency of spontaneous mitotic re- detection of gene conversion, crossing over, and SSA. The cassette consists of combination was reported to increase in the pancreas, but not in two duplicated copies of GFP-Pem1 as described in Fig. 1A, but the second the skin, of mice carrying a fluorescent reporter cassette (29). copy contains full-length GFP-Pem1. (B) Reporter cassette HRIF for detection Spontaneous mitotic recombination is a rare event likely trig- of gene conversion and crossing over. This cassette differs from HRF in that gered by a spontaneous DSB. We hypothesize that as the fre- the second copy of GFP-Pem1 is placed in front of the copy containing I-SceI quency of DSBs increases with age the spontaneous HR events sites, disabling SSA. (C) Rad52 overexpression stimulates SSA. HRF and HRIF are recorded more frequently despite an overall decline in HR were integrated in HCA2 cells. SSA efficiency was calculated as HRF minus Drosophila μ repair. Similarly, in , the frequency of ectopic re- HRIF. To validate this method of measuring SSA, 5 g of the Rad52 expres- fl sion vector were cotransfected with 0.1 μg of pDsRed2-N1 and I-SceI ex- combination increases with age (30), which may re ect a de- MEDICAL SCIENCES pression vector into reporter cells at PD52. (D) SIRT6 overexpression does not crease in accurate intersister gene conversion events, the type of stimulate SSA. Cells were cotransfected with 5 μg of I-SceI vector and 0.1 μg repair measured by our assay. of pDsRed2-N1. Four days after transfection cells were analyzed by FACS, We found that the levels of several HR proteins decline with and the GFP+/DsRed+ ratio was used as the measure of HR efficiency. All replicative age. Rad51 showed the strongest decline with rep- experiments were repeated at least three times and error bars show SDs. licative age, and supplementing it rescued repair in middle- agedcellsbutfailedtodosoinpresenescentcells,indicating that past a certain age, additional Rad51 is no longer capable measures repair events mediated by the HR pathway. of helping repair. Rad52 was inhibitory, which likely reflects A challenge of analyzing HR repair in the context of replicative the role of Rad52 in the error-prone SSA pathway (31). We senescence is that reporter constructs must be integrated in hypothesize that increased levels of Rad52 channeled repair primary human cells, and by the time integrants are selected, toward SSA. Supplementing Rad51, Rad51C, and NBS1, either cells exhaust their replicative potential. We overcame this alone or in combination, did not lead to a rescue of the HR problem by pooling integrating clones at low PD numbers. An- defect in presenescent cells. Why did they fail to rescue HR other advantage of this approach is that clones with multiple repair? All of these proteins could be classified as enzymes integration sites are combined, removing a potential bias at- directly involved in the HR reaction. Because these proteins tributable to position effect. work in a complex, it may be difficult to obtain ideal ratios fi HR ef ciency declined sharply with replicative age and was when overexpressing them. Furthermore, supplementation of > almost completely abolished at PDs 60. The reduction in HR additional components of the HR complex may be required. repair was not explained by cell cycle arrest in the older cells A better solution than supplementing downstream factors because the presenescent cells were actively dividing and had would be altering an upstream regulator of HR. Indeed, a similar ratio of S/G2/M compared with the younger cells. Thus, overexpressing SIRT6 stimulated HR in both middle-aged and cellular aging is intrinsically associated with decline in DNA- presenescent cells. This was the only treatment to work in repair capacity. presenescent cells. Furthermore, we showed that stimulation of Our earlier work demonstrated that DNA repair by NHEJ DSB repair in aged cells depends on mono-ADP ribosylation becomes less efficient and more error-prone with replicative activity of SIRT6 and requires PARP1. We propose that senescence (16). NHEJ in presenescent cells resulted in large SIRT6 serves as an upstream regulator of HR repair, by con- deletions. The reduction in NHEJ efficiency was approximately trolling PARP1 via mono-ADP ribosylation (32), and modu- threefold between young and presenescent cells (16), which is lating SIRT6 activity can overcome the senescence-related a much smaller change compared with 38-fold reduction in the decline in multiple downstream repair factors. Our results efficiency of HR repair. Taken together, these observations paint suggest that SIRT6 promotes recruitment of DNA-repair the following picture of the DSB repair in aging cells. The pre- proteins to DNA lesions. We show here that SIRT6 stimulates cise HR repair is strongly repressed, leaving the DSBs to be recruitment of Rad51, and our earlier report (11) showed that repaired by an error-prone NHEJ pathway. The NHEJ pathway SIRT6 accelerates recruitment of NBS1. In addition, SIRT6 itself becomes less efficient and prone to larger deletions, leading may regulate early steps in the HR pathway that involve strand to mutations and the loss of genetic information. Decline in DSB resection because it also stimulates alternative NHEJ (11) and repair capacity in senescing cells may be directly relevant to strand resection mediated by CtIP (12). Interestingly, SIRT6 human aging. Aging tissues accumulate senescent and pre- did not stimulate the error-prone SSA pathway, suggesting that senescent cells (20–26); thus, the landscape of DNA repair may it may bias DSB repair toward less mutagenic, precise gene change with age. Inefficient and error-prone repair leads to conversion events.

Mao et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 The accumulation of genomic rearrangements is a hallmark of age-related disease and suggest that constitutive SIRT6 activa- aging and contributes to the loss of tissue functionality and the tion may even delay the onset of age-related pathologies. increased incidence of tumorigenesis that characterize the aging process. Here, we demonstrated that the efficiency of HR repair Methods declines precipitously during cellular aging and that over- Cell Culture. HCA2 cells are primary human foreskin fibroblasts. All cells were ’ expression of the DNA repair factor, SIRT6, was uniquely able grown in a 5% CO2,3%O2 incubatorat37°C,inEagles minimal essential medium supplemented with 15% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. to rescue this decline. It was recently reported that SIRT6 overexpression extends lifespan in mice (33); it is possible that Construction of HCA2-HR Cell Lines. HCA2 cells at PD24, which is the lowest PD protection against genomic instability contributes to this phe- available in our laboratory stock, were transfected with 0.1 μg of HR con- notype. Other members of the sirtuin gene family have been struct linearized by NheI. Cells were kept in media with 1 mg/mL G418 for 8 shown to be amenable to pharmacological stimulation (34, 35), d to select colonies with integrated reporter cassettes. After colonies were and recent reports have suggested that SIRT6 may be an in- formed, cells were trypsinized, reseeded, and cultured in one plate until they reached confluence. Three separate transfections were performed, giving triguing candidate for activation to ameliorate the effect of rise to three independent pools. These cells lines were named HCA2-HR. multiple age-related pathologies. In addition to lifespan exten- HCA2-HR cell lines were then serially passaged, with splits every 3–4 d, until sion, a separate study indicated that SIRT6 overexpression they reached senescence at PD71. protects against diet–induced obesity (36). Additionally, we have Methods for quantitative PCR, plasmids, antibodies, transfections, FACS observed that SIRT6 overexpression is selectively cytotoxic to analyses, measurements of SIRT6 biochemical activities, and immunofluo- multiple cancer cell lines (37), and several studies have suggested rescence are provided in SI Methods. that SIRT6 may function as an agonist of NF-κB–induced in- fl fi ACKNOWLEDGMENTS. We thank Junjie Chen for kindly providing us with ammation (38, 39). Our ndings with regard to DNA repair CtIP plasmid. This work was supported by grants from the National Institutes lend credence to the strategy of activating SIRT6 to alleviate of Health (to V.G.) and the Ellison Medical Foundation (to V.G. and A.S.).

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