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

Abstract

Genomic instability is a hallmark of aging tissues. Genomic instability may arise from the inefficient or aberrant function of DNA double-stranded break (DSB) repair. DSBs are repaired by homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). HR is a precise pathway, whereas NHEJ frequently leads to deletions or insertions at the repair site. Here, we used normal human fibroblasts with a chromosomally integrated HR reporter cassette to examine the changes in HR efficiency as cells progress to replicative senescence. We show that HR declines sharply with increasing replicative age, with an up to 38-fold decrease in efficiency in presenescent cells relative to young cells. This decline is not explained by a reduction of the number of cells in S/G 2 /M stage as presenescent cells are actively dividing. Expression of proteins involved in HR such as Rad51, Rad51C, Rad52, NBS1, and Sirtuin 6 (SIRT6) diminished with cellular senescence. Supplementation of Rad51, Rad51C, Rad52, and NBS1 proteins, either individually or in combination, did not rescue the senescence-related decline of HR. However, overexpression of SIRT6 in "middle-aged" and presenescent cells strongly stimulated HR repair, and this effect was dependent on mono-ADP ribosylation activity of poly(ADP-ribose) polymerase (PARP1). These results suggest that in aging cells, the precise HR pathway becomes repressed giving way to a more error-prone NHEJ pathway. These changes in the processing of DSBs may contribute to age-related genomic instability and a higher incidence of cancer with age. SIRT6 activation provides a potential therapeutic strategy to prevent the decline in genome maintenance.A ging is associated with an increased mutation rate (1) and the appearance of genomic rearrangements (2). The accumulation of mutations and rearrangements is a contributing cause of aging and leads to a decline of tissue functionality and an increased incidence of tumors. These mutations and genomic rearrangements arise from aberrant repair of DNA doublestranded breaks (DSBs).DSBs are dangerous DNA lesions. If left unrepaired or repaired incorrectly, DSBs result in a massive loss of genetic information, chromosomal aberrations, or cell death. DSBs are repaired by two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR) (3). NHEJ modifies the broken DNA ends and ligates them together with no requirement for homology, often generating deletions or insertions (4). In contrast, HR uses an undamaged DNA template to repair the break, leading to the reconstitution of the original sequence (5). HR repair is responsible for approximately one quarter of DNA repair events and has much slower repair kinetics than NHEJ (6). HR repair begins with the 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 are resected, Rad51 forms nucleoprotein filaments and mediates strand invasion of the filament into duplex DNA, usual...

Impaired DNA double-strand break repair contributes to the age-associated rise of genomic instability in humans

“…All cultured cells were counted on a Millipore Muse machine (Hayward, CA, USA) and the PD number was calculated as previously reported. 21,29 Plasmids, siRNAs and antibodies. The backbone vectors overexpressing XRCC4 and DNA Lig4 are based on pEGFP-N1.…”

“…29 In this case, at least 10 000 cells were analyzed on FACS. All the data was further analyzed using the software of FlowJo (Ashland, OR, USA).…”

“…21,29 In presenescent cells, HR efficiency declines by 38-fold, whereas NHEJ changes by only~two to threefold. Consistent with the above results, our current aging study also shows a sharp decline of HR efficiency during aging, with the biggest difference of an~30-fold change, whereas the change of NHEJ with age is relatively mild, albeit statistically significant.…”

SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner

“…15 In presenescent cells, restoration of SIRT6 strongly improves HR in a PARP1 dependent manner. 17 Intriguingly, although the molecular mechanisms remain to be elucidated, SIRT6 was first proposed to be involved in BER. 22 Knocking out SIRT6 in mice leads to a phenotype of progeria.…”

“…Several studies indicate that SIRT6 might execute its anti-aging function by participating in DNA double strand break (DSB) repair. [15][16][17][18][19][20] In response to DNA damage, SIRT6 helps recruit SNF2H, a chromatin remodeler, therefore facilitating the recruitment of 53BP1 and BRCA1 to damage sites. 19 SIRT6 promotes classical nonhomologous end joing (NHEJ) by stabilizing DNAPKcs at damage site.…”