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, 14 (2), 269-76

SIRT6 Rescues the Age Related Decline in Base Excision Repair in a PARP1-dependent Manner

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SIRT6 Rescues the Age Related Decline in Base Excision Repair in a PARP1-dependent Manner

Zhu Xu et al. Cell Cycle.

Abstract

In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.

Keywords: PARP1; SIRT6; SIRTUIN; aging; base excision repair; mono-ADP-ribosylation.

Figures

Figure 1.
Figure 1.
BER efficiency declines with age. (A) Schematic depiction of the plasmid reactivation assay used to analyze BER efficiency. Ten micrograms of pEGFP-N1 were mixed with methylene blue at the indicated concentrations, followed by a 60-minute irradiation treatment with visible light generated by a 100-walt bulb at a distance of 18 cm. Then the damaged pEGFP-N1 vector was purified from the mixture using the Axygen cleanup kit (Axygen, Cat. #AP-PCR-250) before 0.05 μg damaged pEGFP-N1 was transfected to fibroblasts for further FACS analysis. (B) MB+VL treatment damages the expression of EGFP gene in comparison to an untreated control. (C) Validation of the BER analysis assay. 0.05 μg of MB + VL treated pEGFP-N1 together with 0.005 μg of pDsRed2-N1, for normalizing differences in transfection efficiency, was transfected into control or PARP1 depleted HCA2-hTERT cells. Then the ratio of GFP+ cells vs. DsRed+ cells was employed as the measure of BER efficiency. (D) BER efficiency negatively correlates with age. Damaged pEGFP-N1, by MB+VL, and pDsRed2-N1 were co-transfected into foreskin fibroblast cell lines isolated from donors at different ages. When transfections were performed, all cell lines were at a population doubling (PD) number of ∼16. All experiments were repeated at least 3 times. Error bars represent standard deviation (S.D.). ** P < 0.01
Figure 2.
Figure 2.
SIRT6 expression level negatively correlates with age but positively correlates with BER efficiency. (A) Western blot showing changes in BER factor expression levels with age. Nineteen cell lines, isolated from donors of different ages, were split 48 hours before being harvested for protein extract. 50 μg of protein extracts were loaded on SDS-PAGE gels for further Western blot analysis. (B) Expression levels of SIRT6, but not other major BER factors, had a significant negative correlation with age. The expression of various BER factors was quantified with ImageJ software. The quantified data, together with age, was further analyzed with Excel software. (C) Expression level of SIRT6 positively correlates with BER efficiency.
Figure 3.
Figure 3.
SIRT6 is required for repair via the BER pathway. (A) Transient overexpression of SIRT3 and SIRT6 in HCA2-hTERT cells. A vector for expressing SIRT3 or SIRT6 was transfected into HCA2-hTERT cells. At the indicated time points, cells were harvested for Western blot analysis of sirtuin expression. (B) Overexpression of SIRT6 promotes BER efficiency. A pcDNA3.1 control vector or a vector encoding SIRT3 or SIRT6 was co-transfected with damaged pEGFP-N1 and pDsRed2-N1. Seventy-2 hours later, cells were harvested for analysis of BER efficiency using FACSverse. (C) Western blot analysis of mSIRT6 in wild type and SIRT6 knockout MEFs. Exponentially growing MEFs were harvested for to quantify protein expression by Western blot analysis. (D) BER efficiency is reduced in SIRT6 knockout MEFs. 0.05 μg of MB+VL treated pEGFP-N1 and 0.005 μg pDsRed2-N1 were co-transfected into wild type or SIRT6 knockout MEFs 72 hours before cells were harvested for FACS analysis. All the experiments were repeated at least 3 times and error bars represent SD. ** P < 0.01
Figure 4.
Figure 4.
The regulation of BER by SIRT6 is mediated by PARP1. (A) The promotion of the BER pathway by SIRT6 is dependent on both its deacetylase and mono-ADP-ribosyltransferase activities. A control vector or vectors expressing SIRT6 wt or SIRT6 mutants were transfected into HCA2-hTERT cells. Cells were harvested at 24 hours post transfection for Western blot analysis of overexpression. The overexpression effect of SIRT6 wt or mutants on BER was analyzed in the same way as described above, in Figure 3. (B) PJ34 pretreatment abolishes the stimulatory effect of SIRT6 on BER. HCA2-hTERT cells were pretreated with 20 μM PJ34 for 24 hours before transfection with damaged pEGFP-N1 and pDsRed2-N1. After transfection, cells were grown in complete medium supplemented with 20 μM PJ34 for 72 hours before analysis of BER efficiency. (C) SIRT6 fails to stimulate BER in PARP1 depleted HCA2-hTERT cells. A control vector or a SIRT6 expression vector was co-transfected with damaged pEGFP-N1 and pDsRed2-N1 into control HCA2-hTERT or PARP1 depleted HCA2-hTERT. FACS analysis was performed 72 hours later. (D) The K521 site in PARP1 is essential for activation of BER by SIRT6. A control plasmid, PARP1 wt or PARP1 mutant plasmid together with a SIRT6 expression vector were cotransfected into PARP1 depleted HCA2-hTERT cells for analysis of BER efficiency. PARP1 DEEKK represents PARP1 with D387, E488, E491, K498, and K524 mutated to Alanine. All experiments were repeated at least three times. Error bars represent S.D.. ** P < 0.01; * P < 0.05
Figure 5.
Figure 5.
SIRT6 but not XRCC1 or Pol β rescues the decline of BER in old foreskin fibroblast cells. (A) Western blot analysis of XRCC1, Pol β and SIRT6 in the foreskin fibroblasts isolated from a 64 y old donor (FF 64). Vectors expressing the 3 genes were respectively transfected into the cells 24 hours before being harvested for protein extraction. Western blotting was in turn performed with the indicated antibodies. (B) SIRT6 restores the decrease of BER efficiency in the old fibroblasts in PARP1 dependent manner. Vectors expressing XRCC1, Pol β or SIRT6 with damaged pEGFP-N1 and pDsRed2-N1 were cotransfected into fibroblasts isolated from a 26 y and a 64 y old donors (FF26 and F64, respectively). Three days later, cells were harvested for FACS analysis. For PJ34 treatment experiments, PJ34 at 20 μM was supplemented to the complete growth medium for 24 hours before and 72 hours post transfection. The same transfection as Figure 4B was performed in FF64 cells. On day 3 post transfection, cells were harvested for FACS analysis. All the experiments were repeated at least 3 times. Error bars represent S.D.. ** P < 0.01

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