The cGAS-STING signaling pathway is modulated by urolithin A.

During aging, general cellular processes, including autophagic clearance and immunological responses become compromised; therefore, identifying compounds that target these cellular processes is an important approach to improve our health span. The innate immune cGAS-STING pathway has emerged as an important signaling system in the organismal defense against viral and bacterial infections, inflammatory responses to cellular damage, regulation of autophagy, and tumor immunosurveillance. These key functions of the cGAS-STING pathway make it an attractive target for pharmacological intervention in disease treatments and in controlling inflammation and immunity. Here, we show that urolithin A (UA), an ellagic acid metabolite, exerts a profound effect on the expression of STING and enhances cGAS-STING activation and cytosolic DNA clearance in human cell lines. Animal laboratory models and limited human trials have reported no obvious adverse effects of UA administration. Thus, the use of UA alone or in combination with other pharmacological compounds may present a potential therapeutic approach in the treatment of human diseases that involves aberrant activation of the cGAS-STING pathway or accumulation of cytosolic DNA and this warrants further investigation in relevant transgenic animal models.

A POLD3/BLM dependent pathway handles DSBs in transcribed chromatin upon excessive RNA:DNA hybrid accumulation.

Transcriptionally active loci are particularly prone to breakage and mounting evidence suggests that DNA Double-Strand Breaks arising in active genes are handled by a dedicated repair pathway, Transcription-Coupled DSB Repair (TC-DSBR), that entails R-loop accumulation and dissolution. Here, we uncover a function for the Bloom RecQ DNA helicase (BLM) in TC-DSBR in human cells. BLM is recruited in a transcription dependent-manner at DSBs where it fosters resection, RAD51 binding and accurate Homologous Recombination repair. However, in an R-loop dissolution-deficient background, we find that BLM promotes cell death. We report that upon excessive RNA:DNA hybrid accumulation, DNA synthesis is enhanced at DSBs, in a manner that depends on BLM and POLD3. Altogether our work unveils a role for BLM at DSBs in active chromatin, and highlights the toxic potential of RNA:DNA hybrids that accumulate at transcription-associated DSBs.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

Associations of subjective vitality with DNA damage, cardiovascular risk factors and physical performance.

AIM: To examine associations of DNA damage, cardiovascular risk factors and physical performance with vitality, in middle-aged men. We also sought to elucidate underlying factors of physical performance by comparing physical performance parameters to DNA damage parameters and cardiovascular risk factors. METHODS: We studied 2487 participants from the Metropolit cohort of 11 532 men born in 1953 in the Copenhagen Metropolitan area. The vitality level was estimated using the SF-36 vitality scale. Cardiovascular risk factors were determined by body mass index (BMI), and haematological biochemistry tests obtained from non-fasting participants. DNA damage parameters were measured in peripheral blood mononuclear cells (PBMCs) from as many participants as possible from a representative subset of 207 participants. RESULTS: Vitality was inversely associated with spontaneous DNA breaks (measured by comet assay) (P = 0.046) and BMI (P = 0.002), and positively associated with all of the physical performance parameters (all P < 0.001). Also, we found several associations between physical performance parameters and cardiovascular risk factors. In addition, the load of short telomeres was inversely associated with maximum jump force (P = 0.018), with lowered significance after exclusion of either arthritis sufferers (P = 0.035) or smokers (P = 0.031). CONCLUSION: Here, we show that self-reported vitality is associated with DNA breaks, BMI and objective (measured) physical performance in a cohort of middle-aged men. Several other associations in this study verify clinical observations in medical practice. In addition, the load of short telomeres may be linked to peak performance in certain musculoskeletal activities.

Senescence induced by RECQL4 dysfunction contributes to Rothmund-Thomson syndrome features in mice.

Cellular senescence refers to irreversible growth arrest of primary eukaryotic cells, a process thought to contribute to aging-related degeneration and disease. Deficiency of RecQ helicase RECQL4 leads to Rothmund-Thomson syndrome (RTS), and we have investigated whether senescence is involved using cellular approaches and a mouse model. We first systematically investigated whether depletion of RECQL4 and the other four human RecQ helicases, BLM, WRN, RECQL1 and RECQL5, impacts the proliferative potential of human primary fibroblasts. BLM-, WRN- and RECQL4-depleted cells display increased staining of senescence-associated ß-galactosidase (SA-ß-gal), higher expression of p16(INK4a) or/and p21(WAF1) and accumulated persistent DNA damage foci. These features were less frequent in RECQL1- and RECQL5-depleted cells. We have mapped the region in RECQL4 that prevents cellular senescence to its N-terminal region and helicase domain. We further investigated senescence features in an RTS mouse model, Recql4-deficient mice (Recql4(HD)). Tail fibroblasts from Recql4(HD) showed increased SA-ß-gal staining and increased DNA damage foci. We also identified sparser tail hair and fewer blood cells in Recql4(HD) mice accompanied with increased senescence in tail hair follicles and in bone marrow cells. In conclusion, dysfunction of RECQL4 increases DNA damage and triggers premature senescence in both human and mouse cells, which may contribute to symptoms in RTS patients.

Impact of DNA polymorphisms in key DNA base excision repair proteins on cancer risk.

Genetic variation in DNA repair genes can modulate DNA repair capacity and may be related to cancer risk. However, study findings have been inconsistent. Inheritance of variant DNA repair genes is believed to influence individual susceptibility to the development of environmental cancer. Reliable knowledge on which the base excision repair (BER) sequence variants are associated with cancer risk would help elucidate the mechanism of cancer. Given that most of the previous studies had inadequate statistical power, we have conducted a systematic review on sequence variants in three important BER proteins. Here, we review published studies on the association between polymorphism in candidate BER genes and cancer risk. We focused on three key BER genes: 8-oxoguanine DNA glycosylase (OGG1), apurinic/apyrimidinic endonuclease (APE1/APEX1) and x-ray repair cross-complementing group 1 (XRCC1). These specific DNA repair genes were selected because of their critical role in maintaining genome integrity and, based on previous studies, suggesting that single-nucleotide polymorphisms (SNPs) in these genes have protective or deleterious effects on cancer risk. A total of 136 articles in the December 13, 2010 MEDLINE database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/pubmed/) reporting polymorphism in OGG1, XRCC1 or APE1 genes were analyzed. Many of the reported SNPs had diverse association with specific human cancers. For example, there was a positive association between the OGG1 Ser326Cys variant and gastric and lung cancer, while the XRCC1 Arg399Gln variant was associated with reduced cancer risk. Gene-environment interactions have been noted and may be important for colorectal and lung cancer risk and possibly other human cancers.

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms.

Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

Role of mitochondrial hOGG1 and aconitase in oxidant-induced lung epithelial cell apoptosis.

8-Oxoguanine DNA glycosylase (Ogg1) repairs 8-oxo-7,8-dihydroxyguanine (8-oxoG), one of the most abundant DNA adducts caused by oxidative stress. In the mitochondria, Ogg1 is thought to prevent activation of the intrinsic apoptotic pathway in response to oxidative stress by augmenting DNA repair. However, the predominance of the beta-Ogg1 isoform, which lacks 8-oxoG DNA glycosylase activity, suggests that mitochondrial Ogg1 functions in a role independent of DNA repair. We report here that overexpression of mitochondria-targeted human alpha-hOgg1 (mt-hOgg1) in human lung adenocarcinoma cells with some alveolar epithelial cell characteristics (A549 cells) prevents oxidant-induced mitochondrial dysfunction and apoptosis by preserving mitochondrial aconitase. Importantly, mitochondrial alpha-hOgg1 mutants lacking 8-oxoG DNA repair activity were as effective as wild-type mt-hOgg1 in preventing oxidant-induced caspase-9 activation, reductions in mitochondrial aconitase, and apoptosis, suggesting that the protective effects of mt-hOgg1 occur independent of DNA repair. Notably, wild-type and mutant mt-hOgg1 coprecipitate with mitochondrial aconitase. Furthermore, overexpression of mitochondrial aconitase abolishes oxidant-induced apoptosis whereas hOgg1 silencing using shRNA reduces mitochondrial aconitase and augments apoptosis. These findings suggest a novel mechanism that mt-hOgg1 acts as a mitochondrial aconitase chaperone protein to prevent oxidant-mediated mitochondrial dysfunction and apoptosis that might be important in the molecular events underlying oxidant-induced toxicity.

Bloom's syndrome workshop focuses on the functional specificities of RecQ helicases.

Human cells express five DNA helicases that are paralogs of Escherichia coli RecQ and which constitute the family of human RecQ helicases. Disease-causing mutations in three of these five human DNA helicases, BLM, WRN, and RECQL4, cause rare severe human genetic diseases with distinct clinical phenotypes characterized by developmental defects, skin abnormalities, genomic instability, and cancer susceptibility. Although biochemical and genetic evidence support roles for all five human RecQ helicases in DNA replication, DNA recombination, and the biological responses to DNA damage, many questions concerning the various functions of the human RecQ helicases remain unanswered. Researchers investigating human and non-human RecQ helicases held a workshop on May 27-28, 2008, at the University of Chicago Gleacher Center, during which they shared insights, discussed recent progress in understanding the biochemistry, biology, and genetics of the RecQ helicases, and developed research strategies that might lead to therapeutic approaches to the human diseases that result from mutations in RecQ helicase genes. Some workshop sessions were held jointly with members of a recently formed advocacy and support group for persons with Bloom's syndrome and their families. This report describes the outcomes and main discussion points of the workshop.

DNA repair, mitochondria, and neurodegeneration.

Accumulation of nuclear and mitochondrial DNA damage is thought to be particularly deleterious in post-mitotic cells, which cannot be replaced through cell division. Recent experimental evidence demonstrates the importance of DNA damage responses for neuronal survival. Here, we summarize current literature on DNA damage responses in the mammalian CNS in aging and neurodegeneration. Base excision repair (BER) is the main pathway for the removal of small DNA base modifications, such as alkylation, deamination and oxidation, which are generated as by-products of normal metabolism and accumulate with age in various experimental models. Using neuronal cell cultures, human brain tissue and animal models, we and others have shown an active BER pathway functioning in the brain, both in the mitochondrial and nuclear compartments. Mitochondrial DNA repair may play a more essential role in neuronal cells because these cells depend largely on intact mitochondrial function for energy metabolism. We have characterized several BER enzymes in mammalian mitochondria and have shown that BER activities change with age in mitochondria from different brain regions. Together, the results reviewed here advocate that mitochondrial DNA damage response plays an important role in aging and in the pathogenesis of neurodegenerative diseases.

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage.

The Werner syndrome protein (WRN) and chromatin assembly factor 1 (CAF-1) are both involved in the maintenance of genome stability. In response to DNA-damaging signals, both of these proteins relocate to sites where DNA synthesis occurs. However, the interaction between WRN and CAF-1 has not yet been investigated. In this report, we show that WRN interacts physically with the largest subunit of CAF-1, hp150, in vitro and in vivo. Although hp150 does not alter WRN catalytic activities in vitro, and the chromatin assembly activity of CAF-1 is not affected in the absence of WRN in vivo, this interaction may have an important role during the cellular response to DNA replication fork blockage and/or DNA damage signals. In hp150 RNA-mediated interference (RNAi) knockdown cells, WRN partially formed foci following hydroxyurea (HU) treatment. However, in the absence of WRN, hp150 did not relocate to form foci following exposure to HU and ultraviolet light. Thus, our results demonstrate that WRN responds to DNA damage before CAF-1 and suggest that WRN may recruit CAF-1, via interaction with hp150, to DNA damage sites during DNA synthesis.

Redundancy of DNA helicases in p53-mediated apoptosis.

A subset of DNA helicases, the RecQ family, has been found to be associated with the p53-mediated apoptotic pathway and is involved in maintaining genomic integrity. This family contains the BLM and WRN helicases, in which germline mutations are responsible for Bloom and Werner syndromes, respectively. TFIIH DNA helicases, XPB and XPD, are also components in this apoptotic pathway. We hypothesized that there may be some redundancy between helicases in their ability to complement the attenuated p53-mediated apoptotic levels seen in cells from individuals with diseases associated with these defective helicase genes. The attenuated apoptotic phenotype in Bloom syndrome cells was rescued not only by ectopic expression of BLM, but also by WRN or XPB, both 3' --> 5' helicases, but not expression of the 5' --> 3' helicase XPD. Overexpression of Sgs1, a WRN/BLM yeast homolog, corrected the reduction in BS cells only, which is consistent with Sgs1 being evolutionarily most homologous to BLM. A restoration of apoptotic levels in cells from WS, XPB or XPD patients was attained only by overexpression of the specific helicase. Our data suggest a limited redundancy in the pathways of these RecQ helicases in p53-induced apoptosis.

Localization of mitochondrial DNA base excision repair to an inner membrane-associated particulate fraction.

Mitochondrial DNA (mtDNA) contains high levels of oxidative damage relative to nuclear DNA. A full, functional DNA base excision repair (BER) pathway is present in mitochondria, to repair oxidative DNA lesions. However, little is known about the organization of this pathway within mitochondria. Here, we provide evidence that the mitochondrial BER proteins are not freely soluble, but strongly associated with an inner membrane-containing particulate fraction. Uracil DNA glycosylase, oxoguanine DNA glycosylase and DNA polymerase gamma activities all co-sedimented with this particulate fraction and were not dissociated from it by detergent (0.1% or 1.0% NP40) treatment. The particulate associations of these activities were not due to their binding mtDNA, which is itself associated with the inner membrane, as they also localized to the particulate fraction of mitochondria from 143B (TK-) rho(0) cells, which lack mtDNA. However, all of the BER activities were at least partially solubilized from the particulate fraction by treatment with 150-300 mM NaCl, suggesting that electrostatic interactions are involved in the association. The biological implications of the apparent immobilization of BER proteins are discussed.

No evidence of mitochondrial respiratory dysfunction in OGG1-null mice deficient in removal of 8-oxodeoxyguanine from mitochondrial DNA.

Accumulation of high levels of mutagenic oxidative mitochondrial DNA (mtDNA) lesions like 8-oxodeoxyguanine (8-oxodG) is thought to be involved in the development of mitochondrial dysfunction in aging and in disorders associated with aging. Mice null for oxoguanine DNA glycosylase (OGG1) are deficient in 8-oxodG removal and accumulate 8-oxodG in mtDNA to levels 20-fold higher than in wild-type mice (N.C. Souza-Pinto et al., 2001, Cancer Res. 61, 5378-5381). We have used these animals to investigate the effects on mitochondrial function of accumulating this particular oxidative base modification. Despite the presence of high levels of 8-oxodG, mitochondria isolated from livers and hearts of Ogg1-/- mice were functionally normal. No differences were detected in maximal (chemically uncoupled) respiration rates, ADP phosphorylating respiration rates, or nonphosphorylating rates with glutamate/malate or with succinate/rotenone. Similarly, maximal activities of respiratory complexes I and IV from liver and heart were not different between wild-type and Ogg1-/- mice. In addition, there was no indication of increased oxidative stress in mitochondria from Ogg1-/- mice, as measured by mitochondrial protein carbonyl content. We conclude, therefore, that highly elevated levels of 8-oxodG in mtDNA do not cause mitochondrial respiratory dysfunction in mice.

The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity.

The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; alpha-Ogg1, which mainly localizes to the nucleus and beta-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from rho(0) cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that beta-Ogg1 protein levels are approximately 80% reduced in rho(0) cells, suggesting beta-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant beta-Ogg1. Surprisingly, recombinant beta-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since beta-Ogg1 lacks the C-terminal alphaO helix present in alpha-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317-323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the alphaO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why beta-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of alpha-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that alpha-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. beta-Ogg1 may play a novel role in human mitochondria.

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA.

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK(-) osteosarcoma cells that had been depleted of mtDNA (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(0) cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(0) cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.

Mitochondrial and nuclear DNA base excision repair are affected differently by caloric restriction.

Aging is strongly correlated with the accumulation of oxidative damage in DNA, particularly in mitochondria. Oxidative damage to both mitochondrial and nuclear DNA is repaired by the base excision repair (BER) pathway. The "mitochondrial theory of aging" suggests that aging results from declining mitochondrial function, due to high loads of damage and mutation in mitochondrial DNA (mtDNA). Restriction of caloric intake is the only intervention so far proven to slow the aging rate. However, the molecular mechanisms underlying such effects are still unclear. We used caloric-restricted (CR) mice to investigate whether lifespan extension is associated with changes in mitochondrial BER activities. Mice were divided into two groups, receiving 100% (PF) or 60% (CR) of normal caloric intake, a regime that extends mean lifespan by approximately 40% in CR mice. Mitochondria isolated from CR mice had slightly higher uracil (UDG) and oxoguanine DNA glycosylase (OGG1) activities but marginally lower abasic endonuclease and polymerase gamma gap-filling activities, although these differences were tissue-specific. Uracil-initiated BER synthesis incorporation activities were significantly lower in brain and kidney from CR mice but marginally enhanced in liver. However, nuclear repair synthesis activities were increased by CR, indicating differential regulation of BER in the two compartments. The results indicate that a general up-regulation of mitochondrial BER does not occur in CR.

Human diseases deficient in RecQ helicases.

RecQ helicases are conserved from bacteria to man. Mutations in three of the human RecQ family members give rise to genetic disorders characterized by genomic instability and a predisposition to cancer. RecQ helicases are therefore caretakers of the genome, and although they do not directly regulate tumorigenesis, they influence stability and the rate of accumulation of genetic alterations, which in turn, result in tumorigenesis. Maintenance of genome stability by RecQ helicases likely involves their participation in DNA replication, recombination, and repair pathways.

DNA damage and its processing. relation to human disease.

We are constantly exposed to sources of agents that directly damage the genetic material. This exposure comes from environmental sources but also from within our own organisms. DNA damage occurs at a high frequency due to metabolic processes and environmental factors such as various exposures and the intake of food and drugs. The stability and correct function of the DNA is necessary for normal cellular functions and there is good evidence that damage to the DNA can lead to cellular dysfunction, cancer and other diseases, or cell death. To avoid or minimize the damage to DNA we have evolved an elaborate set of DNA repair pathways that survey the DNA and fix the errors. There are several human diseases that are known to be defective in these repair pathways, and the accumulation of DNA damage with time in their genome may then be the cause of the associated high incidence of cancer or of an expedited ageing process. The prevention and/or repair of DNA damage thus represent major concerns in biology and medicine.