Tier 3 — preclinical

JNK phosphorylates SIRT6 to stimulate DNA double-strand break repair in response to oxidative stress by recruiting PARP1 to DNA breaks

Michael Van Meter, Matthew Simon, Gregory Tombline, Alfred May, Timothy D. Morello, Basil P. Hubbard, Katie Bredbenner, Rosa Park, David A. Sinclair, Vilhelm A. Bohr, Vera Gorbunova, Andrei Seluanov

Cell Reports 2016 Volume 16, issue 10, pages 2641–2650

Bibliography

DOI: 10.1016/j.celrep.2016.08.006
PubMed: PMID 27568560
PubMed Central: PMC5089070
Funding: Intramural program of the US National Institute on Aging to V.A.B. Grants from the US National Institute on Aging to M.V.M., V.G., and A.S. Life Extension Foundation to V.G. and A.S.
Competing interests: Not explicitly stated in the published manuscript. Senior author D.A.S. is separately known to have commercial affiliations in the longevity space; readers evaluating the broader body of work from these authors should verify current disclosures.

Study snapshot

Design	Multi-modal mechanistic study. Chromosomally integrated non-homologous end joining and homologous recombination reporter assays in HCA2-hTERT human fibroblasts. Chemical inhibitor and siRNA screen against canonical stress response kinases. In vitro kinase assay with purified JNK and recombinant SIRT6. Phospho-specific antibody generation against SIRT6-pS10. SIRT6 S10A and S10E point mutants tested in SIRT6-knockout mouse embryonic fibroblasts. Laser-induced DNA damage confocal microscopy of GFP-tagged SIRT6 and PARP1 recruitment kinetics.
Model	HCA2-hTERT immortalised normal human diploid foreskin fibroblasts (wild-type, NHEJ reporter, HR reporter). Wild-type, SIRT6-knockout, and PARP1-knockout mouse embryonic fibroblasts on 129 genetic background. U2OS human osteosarcoma cells for laser irradiation. CEMT human T-cell lymphoblasts for endogenous SIRT6 validation.
Sample	Independent biological replicates ranging from three to six per condition. Flow cytometry: minimum 50,000 cells analysed per treatment condition. Confocal microscopy: minimum eight cells per condition analysed for recruitment kinetics. Comet assay: 100 cells scored per independent experiment.
Intervention	Oxidative stress induction with 1 mM paraquat for 16 hours. JNK inhibition with 20 micromolar SP600125 or JNK1/2-targeting siRNAs. Overexpression of wild-type, S10A, or S10E SIRT6. Comparison of chemical inhibitor and genetic perturbation across the p38, ERK, mTOR, AKT, CDK, Raf, and JNK signalling axes.
Duration	Paraquat pretreatment 16 hours. JNK inhibitor pretreatment 2 hours. NHEJ and HR reporter readout 4 days after transfection. Laser-induced damage kinetics measured over 2 to 10 minutes.
Endpoints	Non-homologous end joining efficiency (GFP-positive over DsRed-positive cell ratio); Homologous recombination efficiency (GFP-positive over DsRed-positive cell ratio); Clearance kinetics of gamma-H2AX foci; Comet assay tail moments; SIRT6 recruitment amplitude to laser-induced DNA damage sites; PARP1 recruitment amplitude to laser-induced DNA damage sites; In vitro SIRT6 mono-ADP-ribosylation of PARP1; In vitro PARP1 poly-ADP-ribosylation activity

What the study showed, in plain terms

This is a mechanistic study in human and mouse cells, not a supplement trial or an animal lifespan experiment. The Rochester and Harvard teams behind it were trying to answer a very specific question: how does the cell tell SIRT6 to come to the site of a DNA break when oxidative stress hits?

The answer they uncovered runs through a stress-activated enzyme called c-Jun N-terminal kinase, or JNK. Under oxidative stress, JNK adds a phosphate tag to SIRT6 at position serine 10. This single chemical modification does three connected things: it accelerates SIRT6's arrival at DNA break sites, it stimulates SIRT6's ability to modify a partner protein called PARP1 at a specific site (lysine 521), and it enables efficient recruitment of PARP1 itself to the break — where PARP1 then orchestrates the repair.

To prove this, the team ran a screen against every canonical stress signalling pathway (JNK, p38, ERK, mTOR, AKT, CDK, Raf) and only JNK inhibition blocked SIRT6's ability to promote DNA repair. They generated a phosphorylation-blocking SIRT6 mutant (S10A) that failed to stimulate DNA repair even in cells lacking any other SIRT6, and a phosphorylation-mimicking mutant (S10E) that stimulated DNA repair even without stress. They generated a custom antibody that specifically recognises SIRT6 phosphorylated at S10 and showed the modification rises with paraquat treatment and falls with JNK inhibition.

The finding matters for the ageing story because it provides a molecular explanation for a hormetic response — the phenomenon by which mild stress ends up strengthening a cell's repair capacity. Under oxidative stress, JNK activates SIRT6, SIRT6 activates PARP1, and PARP1 orchestrates DNA break repair. Interfering with this axis, as we do when we chronically inhibit inflammation or oxidative signalling, may unintentionally weaken the DNA repair response.

Key findings

A screen of chemical inhibitors against canonical stress response kinases (JNK, p38, ERK, mTOR, AKT, CDK, Raf) identified JNK as the sole kinase whose inhibition abrogated SIRT6's ability to stimulate DNA double-strand break repair after paraquat-induced oxidative stress. siRNA-mediated JNK knockdown reproduced the effect.
Purified activated JNK directly phosphorylated recombinant SIRT6 in an in vitro kinase reaction, and JNK auto-phosphorylation was reduced in the presence of SIRT6 — indicating SIRT6 is a preferred JNK substrate.
Site-directed mutagenesis of the five reported SIRT6 phosphorylation sites (S10, T294, S303, S330, S338) demonstrated that only S10 phosphorylation was required for stimulation of DNA repair under oxidative stress. S10A phospho-null SIRT6 completely failed to stimulate NHEJ in SIRT6-knockout mouse embryonic fibroblasts; S10E phospho-mimetic SIRT6 constitutively stimulated NHEJ even without oxidative stress.
A custom-generated phospho-specific antibody (Rb5159) demonstrated a 15,000-fold specificity for SIRT6-pS10 over unphosphorylated SIRT6. Paraquat treatment induced S10 phosphorylation in cultured human fibroblasts; JNK inhibition abolished this phosphorylation. Lambda phosphatase treatment confirmed the antibody specificity.
Confocal microscopy of laser-induced DNA damage showed that SIRT6-GFP was rapidly recruited to break sites in a JNK-dependent manner. Wild-type SIRT6 recruited efficiently; S10E SIRT6 recruited more efficiently; S10A SIRT6 recruited poorly.
S10E phospho-mimetic SIRT6 more robustly mono-ADP-ribosylated PARP1 in vitro and produced correspondingly higher PARP1 poly-ADP-ribosylation activity compared with wild-type or S10A SIRT6. S10A and S10E mutations did not affect SIRT6 deacetylation activity on H3K9ac, indicating that the mono-ADP-ribosylation arm of SIRT6, not the deacetylation arm, mediates the DNA repair stimulation.
In SIRT6-knockout mouse embryonic fibroblasts, PARP1-GFP failed to efficiently recruit to laser-induced DNA breaks — placing SIRT6 upstream of PARP1 in the DNA damage response cascade. Mutation of the SIRT6 mono-ADP-ribosylation target site on PARP1 (K521A) similarly prevented PARP1 recruitment to breaks.

What this study can and cannot tell us

A 2026 corrigendum was issued for this paper. Published in Cell Reports on 27 January 2026 (volume 45 issue 1, page 116960, DOI 10.1016/j.celrep.2026.116960, PMID 41548218), the corrigendum notes that the anti-histone-H3 loading control immunoblot shown in Figure 2C was inadvertently duplicated from a separate project during manuscript preparation. Because the experiments were performed more than ten years earlier and the original image files could not be located under the authors' institutional data-retention policy, the immunoblot images in Figure 2C were removed rather than replaced. The corrigendum states that the article's central conclusions are unchanged. Readers relying on this paper for the S10A and S10E expression data specifically should treat that Western blot loading control as retracted while accepting the broader mutational, kinetic, and microscopy findings as valid.

The study is entirely cellular and biochemical. No animal or human physiological outcomes are measured. Whether the JNK-SIRT6-PARP1 axis operates identically in vivo, whether it responds to physiological rather than pharmacological oxidative stress, and whether it produces the healthspan and lifespan effects hypothesised in the discussion remain open questions.

Oxidative stress is induced pharmacologically with paraquat at 1 millimolar for 16 hours. This is a strong and non-physiological stressor. The relevance of the specific S10-JNK response to the milder oxidative fluctuations that occur naturally during ageing, exercise, or dietary variation is not directly tested.

The claim that S10 phosphorylation drives PARP1 recruitment via SIRT6 mono-ADP-ribosylation rests partly on in vitro kinase and ribosylation reactions with purified proteins. The extent to which the same mechanism operates on native chromatin substrates in vivo, and whether other post-translational modifications on SIRT6 (S303, S330, S338, or as-yet-unmapped sites) contribute additively or synergistically, is not resolved.