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

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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 C D E 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 pre- senescent (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 ef- ficiency 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.
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