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SUMMARYThere is currently no established intervention for Cockayne syndrome, a disease characterized by progressive early onset neurodegeneration with features of premature aging. Here, we tested if acetyl-CoA precursors, citrate and beta-hydroxybutyrate, could reduce features of Cockayne syndrome in three model systems. We identified the gene Helicase 89B as a homologue of CSB in drosophila and found that the ketone beta-hydroxybutyrate rescued features of premature aging in Hel89B deficient flies. In mammals, loss of the citrate carrier Indy exacerbated the phenotype of Csbm/m mice which was rescued by a ketogenic diet. The rescue effect appeared to be mediated through ketone stimulated histone acetylation and facilitation of transcriptional readthrough of secondary DNA structures. These findings link a ketogenic diet with transcriptional resolution of secondary structures and DNA repair.

PURPOSE:To enhance classification of variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes in the cancer predisposition Lynch syndrome, we developed the cell-free in vitro MMR activity (CIMRA) assay. Here, we calibrate and validate the assay, enabling its integration with in silico and clinical data.METHODS:Two sets of previously classified MLH1 and MSH2 variants were selected from a curated MMR gene database, and their biochemical activity determined by the CIMRA assay. The assay was calibrated by regression analysis followed by symmetric cross-validation and Bayesian integration with in silico predictions of pathogenicity. CIMRA assay reproducibility was assessed in four laboratories.RESULTS:Concordance between the training runs met our prespecified validation criterion. The CIMRA assay alone correctly classified 65% of variants, with only 3% discordant classification. Bayesian integration with in silico predictions of pathogenicity increased the proportion of correctly classified variants to 87%, without changing the discordance rate. Interlaboratory results were highly reproducible.CONCLUSION:The CIMRA assay accurately predicts pathogenic and benign MMR gene variants. Quantitative combination of assay results with in silico analysis correctly classified the majority of variants. Using this calibration, CIMRA assay results can be integrated into the diagnostic algorithm for MMR gene variants.KEYWORDS:Lynch syndrome; assay calibration; functional assay; variant classification; variants of uncertain significance

Age-related macular degeneration (AMD) is a highly prevalent degenerative disease and a leading cause of vision loss worldwide. Evidence for an inflammatory component in the development of AMD exists, yet the exact mechanisms remain unclear. Bisretinoid N-retinylidene-N-retinylethanolamine (A2E) in retinal pigmental epithelial (RPE) cells, and in extracellular deposits constitutes a hallmark of AMD, but its role in the pathology of AMD is elusive. Here, we tested the hypothesis that A2E is responsible for the heightened inflammatory activity in AMD. To this end, we measured ex vivo mRNA expression of the cytokines TNF-α, IL-6, and IL-10 in whole blood samples after stimulation with A2E in a clinical sample of 27 patients with neovascular AMD and 24 patients with geographic atrophy secondary to AMD. Patients’ spouses (n = 30) were included as non-affected controls. After stimulation with A2E, no statistical differences were found in the median expression level of TNF-α, IL-6, IL-10 between the control group, and the neovascular AMD and the geographic atrophy group. Our findings do not support evidence for the hypothesis, that A2E per se contributes to heightened inflammatory activity in AMD.

The Ku70/80 heterodimer binds to DNA ends and attracts other proteins involved in the non-homologous end-joining (NHEJ) pathway of DNA double-strand break repair. We developed a novel assay to measure DNA binding and release kinetics using differences in F&ouml, rster resonance energy transfer (FRET) of the ECFP-Ku70/EYFP-Ku80 heterodimer in soluble and DNA end bound states. We confirmed that the relative binding efficiencies of various DNA substrates (blunt, 3 nucleotide 5&prime, extension, and DNA hairpin) measured in the FRET assay reflected affinities obtained from direct measurements using surface plasmon resonance. The FRET assay was subsequently used to investigate Ku70/80 behavior in the context of a DNA-dependent kinase (DNA-PK) holocomplex. As expected, this complex was much more stable than Ku70/80 alone, and its stability was influenced by DNA-PK phosphorylation status. Interestingly, the Ku80 C-terminal extension contributed to DNA-PK complex stability but was not absolutely required for its formation. The Ku70 C-terminal SAP domain, on the other hand, was required for the stable association of Ku70/80 to DNA ends, but this effect was abrogated in DNA-PK holocomplexes. We conclude that FRET measurements can be used to determine Ku70/80 binding kinetics. The ability to do this in complex mixtures makes this assay particularly useful to study larger NHEJ protein complexes on DNA ends.

Maintenance of genomic integrity involves cellular processes tailored to specific types of DNA damage. Monogenic disorders in DNA-repair pathways lead to a spectrum of clinical phenotypes that are not always correlated with our understanding of the affected repair pathway.

The A-type lamins (lamin A/C), encoded by the LMNA gene, are important structural components of the nuclear lamina. LMNA mutations lead to degenerative disorders known as laminopathies, including the premature aging disease Hutchinson-Gilford progeria syndrome. In addition, altered lamin A/C expression is found in various cancers. Reports indicate that lamin A/C plays a role in DNA double strand break repair, but a role in DNA base excision repair (BER) has not been described. We provide evidence for reduced BER efficiency in lamin A/C-depleted cells (Lmna null MEFs and lamin A/C-knockdown U2OS). The mechanism involves impairment of the APE1 and POLβ BER activities, partly effectuated by associated reduction in poly-ADP-ribose chain formation. Also, Lmna null MEFs displayed reduced expression of several core BER enzymes (PARP1, LIG3 and POLβ). Absence of Lmna led to accumulation of 8-oxoguanine (8-oxoG) lesions, and to an increased frequency of substitution mutations induced by chronic oxidative stress including GC>TA transversions (a fingerprint of 8-oxoG:A mismatches). Collectively, our results provide novel insights into the functional interplay between the nuclear lamina and cellular defenses against oxidative DNA damage, with implications for cancer and aging.

SummaryThe RecQ helicase RECQL4, mutated in Rothmund-Thomson syndrome, regulates genome stability, aging, and cancer. Here, we identify a crucial role for RECQL4 in DNA end resection, which is the initial and an essential step of homologous recombination (HR)-dependent DNA double-strand break repair (DSBR). Depletion of RECQL4 severely reduces HR-mediated repair and 5′ end resection in vivo. RECQL4 physically interacts with MRE11-RAD50-NBS1 (MRN), which senses DSBs and initiates DNA end resection with CtIP. The MRE11 exonuclease regulates the retention of RECQL4 at laser-induced DSBs. RECQL4 also directly interacts with CtIP via its N-terminal domain and promotes CtIP recruitment to the MRN complex at DSBs. Moreover, inactivation of RECQL4’s helicase activity impairs DNA end processing and HR-dependent DSBR without affecting its interaction with MRE11 and CtIP, suggesting an important role for RECQL4’s unwinding activity in the process. Thus, we report that RECQL4 is an important participant in HR-dependent DSBR.

DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other organisms. Here, we characterize the interaction between MMR and a proofreading-deficient allele of the human replicative DNA polymerase delta, PolδD316A;E318A, which has a higher capacity for strand displacement DNA synthesis than wild type Polδ. Human cell lines overexpressing PolδD316A;E318A display a mild mutator phenotype, while nuclear extracts of these cells exhibit reduced MMR activity in vitro, and these defects are complemented by overexpression or addition of exogenous human Exonuclease 1 (EXO1). By contrast, another proofreading-deficient mutant, PolδD515V, which has a weaker strand displacement activity, does not decrease the MMR activity as significantly as PolδD316A;E318A. In addition, PolδD515V does not increase the mutation frequency in MMR-proficient cells. Based on our findings, we propose that the proofreading activity restricts the strand displacement activity of Polδ in MMR. This contributes to maintain the nicks required for EXO1 entry, and in this manner ensures the dominance of the EXO1-dependent MMR pathway.

Neurons are terminally differentiated cells with a high rate of metabolism and multiple biological properties distinct from their undifferentiated precursors. Previous studies showed that nucleotide excision DNA repair is downregulated in postmitotic muscle cells and neurons. Here, we characterize DNA damage susceptibility and base excision DNA repair (BER) capacity in undifferentiated and differentiated human neural cells. The results show that undifferentiated human SH-SY5Y neuroblastoma cells are less sensitive to oxidative damage than their differentiated counterparts, in part because they have robust BER capacity, which is heavily attenuated in postmitotic neurons. The reduction in BER activity in differentiated cells correlates with diminished protein levels of key long patch BER components, flap endonuclease-1, proliferating cell nuclear antigen, and ligase I. Thus, because of their higher BER capacity, proliferative neural progenitor cells are more efficient at repairing DNA damage compared with their neuronally differentiated progeny.

Exonuclease 1 (EXO1) is a multifunctional 5' → 3' exonuclease and a DNA structure-specific DNA endonuclease. EXO1 plays roles in DNA replication, DNA mismatch repair (MMR) and DNA double-stranded break repair (DSBR) in lower and higher eukaryotes and contributes to meiosis, immunoglobulin maturation, and micro-mediated end-joining in higher eukaryotes. In human cells, EXO1 is also thought to play a role in telomere maintenance. Mutations in the human EXO1 gene correlate with increased susceptibility to some cancers. This review summarizes recent studies on the enzymatic functions and biological roles of EXO1, its possible protective role against cancer and aging, and regulation of EXO1 by posttranslational modification.

RECQL4 is one of five members of the human RecQ helicase family, and is implicated in three syndromes displaying accelerating aging, developmental abnormalities and a predisposition to cancer. In this study, we purified three variants of RECQL4 carrying previously reported patient mutations. These three mutant proteins were analyzed for the known biochemical activities of RECQL4: DNA binding, unwinding of duplex DNA, ATP hydrolysis and annealing of simplex DNA. Further, the mutant proteins were evaluated for stability and recruitment to sites of laser-induced DNA damage. One mutant was helicase-dead, had marginal ATPase activity and may be structurally compromised, while the other two showed greatly reduced helicase and ATPase activities. The remaining biochemical activities and ability to recruit to damage sites were not significantly impaired for any of the mutants. Our findings demonstrate a consistent pattern of functional deficiency and provide further support for a helicase-dependent cellular function of RECQL4 in addition to its N-terminus-dependent role in initiation of replication, a function that may underlie the phenotype of RECQL4-linked disease.

Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phase progression and in response to laser-induced DNA double strand breaks (DSBs). We show that hEXO1 and PCNA co-localize in replication foci. This apparent interaction is sustained throughout S-phase. We also demonstrate that hEXO1 is rapidly recruited to DNA DSBs. We have identified a PCNA interacting protein (PIP-box) region on hEXO1 located in its COOH-terminal ((788)QIKLNELW(795)). This motif is essential for PCNA binding and co-localization during S-phase. Recruitment of hEXO1 to DNA DSB sites is dependent on the MMR protein hMLH1. We show that two distinct hMLH1 interaction regions of hEXO1 (residues 390-490 and 787-846) are required to direct the protein to the DNA damage site. Our results reveal that protein domains in hEXO1 in conjunction with specific protein interactions control bi-directional routing of hEXO1 between on-going DNA replication and repair processes in living cells.

Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.

AGING is associated with elevated oxidative stress and DNA damage. To achieve healthy aging, we must begin to understand how diet affects cellular processes. We postulated that fruit-enriched diets might initiate a program of enhanced DNA repair and thereby improve genome integrity. C57Bl/6 J mice were fed for 14 weeks a control diet or a diet with 8% peach or nectarine extract. The activities of DNA repair enzymes, the level of DNA damage, and gene expression changes were measured. Our study showed that repair of various oxidative DNA lesions was more efficient in liver extracts derived from mice fed fruit-enriched diets. In support of these findings, gas chromatography–mass spectrometry analysis revealed that there was a decrease in the levels of formamidopyrimidines in peach-fed mice compared with the controls. Additionally, microarray analysis revealed that NTH1 was upregulated in peach-fed mice. Taken together, these results suggest that an increased intake of fruits might modulate the efficiency of DNA repair, resulting in altered levels of DNA damage.

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1−/–) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1−/– mice were more vulnerable to oxidative insults than were OGG1+/+ neurons, and OGG1−/– mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1+/+ mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1−/– mice compared with OGG1+/+ mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome.

The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 T-antigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite transcriptional orientation in the immunoglobulin (Ig) heavy (H) chain locus between the D and JH segments. SV40 T-antigen expression appeared to result from retention of the targeted germline allele and concomitant antisense transcription of SV40 large T in mature B cells, leading to chronic lymphocytic leukemia (CLL). Although B-cell development was unperturbed in young mice, aging mice showed accumulation of a monoclonal B-cell population in which the targeted IgH allele was in germline configuration and the wild-type IgH allele had a productive V(D)J recombination. These leukemic B cells were IgDlowCD5+ and manifested nonrandom usage of V, D, and J segments. VH regions were either unmutated, with preferential usage of the VH11 family, or manifested extensive somatic hypermutation. Our findings provide an animal model for B-CLL and show that pathways activated by SV40 T antigen play important roles in the pathogenesis of B-CLL.

Repair of DNA double-strand breaks (DSBs) is predominantly mediated by nonhomologous end joining (NHEJ) in mammalian cells. NHEJ requires binding of the Ku70-Ku80 heterodimer (Ku70/80) to the DNA ends and subsequent recruitment of the DNA-dependent protein kinase catalytic subunit (DNA-PK(CS)) and the XRCC4/ligase IV complex. Activation of the DNA-PK(CS) serine/threonine kinase requires an interaction with Ku70/80 and is essential for NHEJ-mediated DSB repair. In contrast to previous models, we found that the carboxy terminus of Ku80 is not absolutely required for the recruitment and activation of DNA-PK(CS) at DSBs, although cells that harbored a carboxy-terminal deletion in the Ku80 gene were sensitive to ionizing radiation and showed reduced end-joining capacity. More detailed analysis of this repair defect showed that DNA-PK(CS) autophosphorylation at Thr2647 was diminished, while Ser2056 was phosphorylated to normal levels. This resulted in severely reduced levels of Artemis nuclease activity in vivo and in vitro. We therefore conclude that the Ku80 carboxy terminus is important to support DNA-PK(CS) autophosphorylation at specific sites, which facilitates DNA end processing by the Artemis endonuclease and the subsequent joining reaction.

Non-homologous end-joining (NHEJ) is an essential DNA double strand break repair pathway during all cell cycle stages. Deficiency in NHEJ factors can lead to accumulation of unrepaired DNA breaks or faulty DNA repair, which may ultimately result in cell death, senescence or carcinogenesis. The Ku70/80 heterodimer is a key-player in the NHEJ pathway and binds to DNA termini with high affinity, where it helps to protect DNA ends from degradation and to recruit other NHEJ factors required for repair. The mechanism of Ku70/80 detachment from the DNA helix after completion of DNA repair is incompletely understood. Some data suggest that certain DNA repair factors are ubiquitylated and targeted for proteasomal degradation after repair. Recent studies suggest that Ku80 is conjugated to lysine48-linked ubiquitin chains by the Skp1-Cullin-F-box (SCF) complex and/or the RING finger protein 8 (RNF8) ubiquitin-protein ligases, followed by rapid proteasomal degradation. In this review we address the structure and function of the Ku70/80 heterodimer and how ubiquitylation may affect the release of Ku70/80 from chromatin and its subsequent degradation via the ubiquitin-proteasome system.

We report biochemical characterization of human full-length EXO1 including thermodynamic stability and flap activity on DNA flap structures. Our results reveal novel mechanistic insights into the processing of flap structures and a possible role of EXO1 in strand displacement., Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5′ flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement.

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. 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.

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.

International audience; RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Cerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a CFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to gamma-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.

We have established a proteome reference map for Medicago truncatula root proteins using two-dimensional gel electrophoresis combined with peptide mass fingerprinting to aid the dissection of nodulation and root developmental pathways by proteome analysis. M. truncatula has been chosen as a model legume for the study of nodulation-related genes and proteins. Over 2,500 root proteins could be displayed reproducibly across an isoelectric focussing range of 4-7. We analysed 485 proteins by peptide mass fingerprinting, and 179 of those were identified by matching against the current M. truncatula expressed sequence tag (EST) database containing DNA sequences of approximately 105,000 ESTs. Matching the EST sequences to available plant DNA sequences by BLAST searches enabled us to predict protein function. The use of the EST database for peptide identification is discussed. The majority of identified proteins were metabolic enzymes and stress response proteins, and 44% of proteins occurred as isoforms, a result that could not have been predicted from sequencing data alone. We identified two nodulins in uninoculated root tissue, supporting evidence for a role of nodulins in normal plant development. This proteome map will be updated continuously (http://semele.anu.edu.au/2d/2d.html) and will be a powerful tool for investigating the molecular mechanisms of root symbioses in legumes.

Base excision repair (BER) is the most prominent DNA repair pathway in human mitochondria. BER also results in a temporary generation of AP-sites, single-strand breaks and nucleotide gaps. Thus, incomplete BER can result in the generation of DNA repair intermediates that can disrupt mitochondrial DNA replication and transcription and generate mutations. We carried out BER analysis in highly purified mitochondrial extracts from human cell lines U2OS and HeLa, and mouse brain using a circular DNA substrate containing a lesion at a specific position. We found that DNA ligation is significantly slower than the preceding mitochondrial BER steps. Overexpression of DNA ligase III in mitochondria improved the rate of overall BER, increased cell survival after menadione induced oxidative stress and reduced autophagy following the inhibition of the mitochondrial electron transport chain complex I by rotenone. Our results suggest that the amount of DNA ligase III in mitochondria may be critical for cell survival following prolonged oxidative stress, and demonstrate a functional link between mitochondrial DNA damage and repair, cell survival upon oxidative stress, and removal of dysfunctional mitochondria by autophagy.

Cockayne Syndrome is a segmental premature aging syndrome, which can be caused by loss of function of the CSB protein. CSB is essential for genome maintenance and has numerous interaction partners with established roles in different DNA repair pathways including transcription coupled nucleotide excision repair and base excision repair. Here, we describe a new interaction partner for CSB, the DNA glycosylase NEIL2. Using both cell extracts and recombinant proteins, CSB and NEIL2 were found to physically interact independently of DNA. We further found that CSB is able to stimulate NEIL2 glycosylase activity on a 5-hydroxyl uracil lesion in a DNA bubble structure substrate in vitro. A novel 4,6-diamino-5-formamidopyrimidine (FapyA) specific incision activity of NEIL2 was also stimulated by CSB. To further elucidate the biological role of the interaction, immunofluorescence studies were performed, showing an increase in cytoplasmic CSB and NEIL2 co-localization after oxidative stress. Additionally, stalling of the progression of the transcription bubble with α-amanitin resulted in increased co-localization of CSB and NEIL2. Finally, CSB knockdown resulted in reduced incision of 8-hydroxyguanine in a DNA bubble structure using whole cell extracts. Taken together, our data supports a biological role for CSB and NEIL2 in transcription associated base excision repair.

Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins are regulatory phosphorserine/threonine binding proteins involved in the control of diverse cellular events, including cell cycle checkpoint and apoptosis signaling. hEXO1 is regulated by post-translation Ser/Thr phosphorylation in a yet not fully clarified manner, but evidently three phosphorylation sites are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding experiments reveal weak affinity of the more selective isoform 14-3-3σ but both 14-3-3 isoforms η and σ significantly stimulate hEXO1 activity, indicating that these regulatory proteins exert a common regulation mode on hEXO1. Results demonstrate that binding involves the phosphorable amino acid S746 in hEXO1 and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate with PCNA in distinct replication foci and respond to DNA double strand breaks (DSBs), indicating that 14-3-3 binding does not involve regulating the subcellular distribution of hEXO1. Altogether, these results suggest that association may be related to regulation of hEXO1 availability during the DNA damage response to plausibly prevent extensive DNA resection at the damage site, as supported by recent studies.

Glutamate, the major excitatory neurotransmitter in the brain, activates receptors coupled to membrane depolarization and Ca(2+) influx that mediates functional responses of neurons including processes such as learning and memory. Here we show that reversible nuclear oxidative DNA damage occurs in cerebral cortical neurons in response to transient glutamate receptor activation using non-toxic physiological levels of glutamate. This DNA damage was prevented by intracellular Ca(2+) chelation, the mitochondrial superoxide dismutase mimetic MnTMPyP (Mn-5,10,15,20-tetra(4-pyridyl)-21H,23H-porphine chloride tetrakis(methochloride)), and blockade of the permeability transition pore. The repair of glutamate-induced DNA damage was associated with increased DNA repair activity and increased mRNA and protein levels of apurinic endonuclease 1 (APE1). APE1 knockdown induced accumulation of oxidative DNA damage after glutamate treatment, suggesting that APE1 is a key repair protein for glutamate-induced DNA damage. A cAMP-response element-binding protein (CREB) binding sequence is present in the Ape1 gene (encodes APE1 protein) promoter and treatment of neurons with a Ca(2+)/calmodulin-dependent kinase inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species-mediated DNA damage that is then rapidly repaired by a mechanism involving Ca(2+)-induced, CREB-mediated APE1 expression. Our findings reveal a previously unknown ability of neurons to efficiently repair oxidative DNA lesions after transient activation of glutamate receptors.

Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production Low vitality (a component of fatigue) in middle-aged and older adults is an important complaint often identified as a symptom of a disease state or side effect of a treatment. No studies to date have investigated the potential link between dysfunctional mitochondrial ATP production and low vitality. Therefore, we measured a number of cellular parameters related to mitochondrial activity in peripheral blood mononuclear cells (PBMCs) isolated from middle-aged men, and tested for association with vitality. These parameters estimate mitochondrial respiration, reactive oxygen species (ROS) production, and deoxyribonucleotide (dNTP) balance in PBMCs. The population was drawn from the Metropolit cohort of men born in 1953. Vitality level was estimated from the Medical Outcomes Study Short Form 36 (SF-36) vitality scale. We found that vitality score had no association with any of the mitochondrial respiration parameters. However, vitality score was inversely associated with cellular ROS production and cellular deoxythymidine triphosphate (dTTP) levels and positively associated with deoxycytidine triphosphate (dCTP) levels. We conclude that self-reported persistent low vitality is not associated with specific aspects of mitochondrial oxidative phosphorylation capacity in PBMCs, but may have other underlying cellular dysfunctions that contribute to dNTP imbalance and altered ROS production.

AIMS: Accurate biomarkers for early diagnosis of Alzheimer's disease (AD) are badly needed. Recent reports suggest that dysfunctional mitochondria and DNA damage are associated with AD development. In this report, we measured various cellular parameters, related to mitochondrial bioenergetics and DNA damage, in peripheral blood mononuclear cells (PBMCs) of AD and control participants, for biomarker discovery.METHODS: PBMCs were isolated from 53 patients with AD of mild to moderate degree and 30 age-matched healthy controls. Tests were performed on the PBMCs from as many of these participants as possible. We measured glycolysis and mitochondrial respiration fluxes using the Seahorse Bioscience flux analyzer, mitochondrial ROS production using flow cytometry, dNTP levels by way of a DNA polymerization assay, DNA strand breaks using the Fluorometric detection of Alkaline DNA Unwinding (FADU) assay, and APE1 incision activity (in cell lysates) on a DNA substrate containing an AP site (to estimate DNA repair efficiency).RESULTS: In the PBMCs of AD patients, we found reduced basal mitochondrial oxygen consumption, reduced proton leak, higher dATP level, and lower AP endonuclease 1 activity, depending on adjustments for gender and/or age. CONCLUSIONS: This study reveals impaired mitochondrial respiration, altered dNTP pools and reduced DNA repair activity in PBMCs of AD patients, thus suggesting that these biochemical activities may be useful as biomarkers for AD. published