Special Issue "Mitochondrial Dysfunction: A Common Trigger in Neurodegenerative and Metabolic Non-Communicable Diseases".

Non-communicable diseases (NCDs) are non-infectious and non-transmissible chronic disorders [...].

Effects of the Rho GTPase-activating toxin CNF1 on fibroblasts derived from Rett syndrome patients: A pilot study.

The bacterial product CNF1, through its action on the Rho GTPases, is emerging as a modulator of crucial signalling pathways involved in selected neurological diseases characterized by mitochondrial dysfunctions. Mitochondrial impairment has been hypothesized to have a key role in paramount mechanisms underlying Rett syndrome (RTT), a severe neurologic rare disorder. CNF1 has been already reported to have beneficial effects in mouse models of RTT. Using human RTT fibroblasts from four patients carrying different mutations, as a reliable disease-in-a-dish model, we explored the cellular and molecular mechanisms, which can underlie the CNF1-induced amelioration of RTT deficits. We found that CNF1 treatment modulates the Rho GTPases activity of RTT fibroblasts and induces a considerable re-organization of the actin cytoskeleton, mainly in stress fibres. Mitochondria of RTT fibroblasts show a hyperfused morphology and CNF1 decreases the mitochondrial mass leaving substantially unaltered the mitochondrial dynamic. From a functional perspective, CNF1 induces mitochondrial membrane potential depolarization and activation of AKT in RTT fibroblasts. Given that mitochondrial quality control is altered in RTT, our results are suggestive of a reactivation of the damaged mitochondria removal via mitophagy restoration. These effects can be at the basis of the beneficial effects of CNF1 in RTT.

Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells Differentiated In Vitro.

After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.

Microbial Biocontrol Agents and Natural Products Act as Salt Stress Mitigators in Lactuca sativa L.

One of the major problems related to climate change is the increase in land area affected by higher salt concentrations and desertification. Finding economically and environmentally friendly sustainable solutions that effectively mitigate salt stress damage to plants is of great importance. In our work, some natural products and microbial biocontrol agents were evaluated for their long-term effectiveness in reducing salt stress in lettuce (Lactuca sativa L. var. romana) plants. Fourteen different treatments applied to soil pots, with and without salt stress, were analyzed using biometric (leaf and root length and width), physiological (chlorophyll and proline content), and morphological (microscopic preparations) techniques and NGS to study the microbial communities in the soil of plants subjected to different treatments. Under our long-term experimental conditions (90 days), the results showed that salt stress negatively affected plant growth. The statistical analysis showed a high variability in the responses of the different biostimulant treatments. Notably, the biocontrol agents Papiliotrema terrestris (strain PT22AV), Bacillus amyloliquefaciens (strain B07), and Rahnella aquatilis (strain 36) can act as salt stress mitigators in L. sativa. These findings suggest that both microbial biocontrol agents and certain natural products hold promise for reducing the adverse effects of salt stress on plants.

The dual nature of DNA damage response in obesity and bariatric surgery-induced weight loss.

This novel study applies targeted functional proteomics to examine tissues and cells obtained from a cohort of individuals with severe obesity who underwent bariatric surgery (BS), using a Reverse-Phase Protein Array (RPPA). In obese individuals, visceral adipose tissue (VAT), but not subcutaneous adipose tissue (SAT), shows activation of DNA damage response (DDR) markers including ATM, ATR, histone H2AX, KAP1, Chk1, and Chk2, alongside senescence markers p16 and p21. Additionally, stress-responsive metabolic markers, such as survivin, mTOR, and PFKFB3, are specifically elevated in VAT, suggesting both cellular stress and metabolic dysregulation. Conversely, peripheral blood mononuclear cells (PBMCs), while exhibiting elevated mTOR and JNK levels, did not present significant changes in DDR or senescence markers. Following BS, unexpected increases in phosphorylated ATM, ATR, and KAP1 levels, but not in Chk1 and Chk2 nor in senescence markers, were observed. This was accompanied by heightened levels of survivin and mTOR, along with improvement in markers of mitochondrial quality and health. This suggests that, following BS, pro-survival pathways involved in cellular adaptation to various stressors and metabolic alterations are activated in circulating PBMCs. Moreover, our findings demonstrate that the DDR has a dual nature. In the case of VAT from individuals with obesity, chronic DDR proves to be harmful, as it is associated with senescence and chronic inflammation. Conversely, after BS, the activation of DDR proteins in PBMCs is associated with a beneficial survival response. This response is characterized by metabolic redesign and improved mitochondrial biogenesis and functionality. This study reveals physiological changes associated with obesity and BS that may aid theragnostic approaches.

The response to DNA damage during differentiation: pathways and consequences.

Damage to genomic DNA triggers a prompt set of signaling events known as the DNA damage response (DDR) which coordinates DNA repair, cell cycle arrest and ultimately cell death or senescence. Although activation of adequate DNA damage signaling and repair systems depends on the type of lesion and the cell-cycle phase in which it occurs, emerging evidence indicates that DNA repair and DDR function differently in different cellular contexts. Depending on the time maintenance and function of a specific cell type the risk of accumulating DNA damage may vary. For instance, damage to stem cells if not repaired can lead to mutation amplification or propagation through the processes of self-renewal and differentiation, respectively, whereas damage to post-mitotic cells can affect mostly tissue homeostasis. Stem cells are therefore expected to address DNA damage differently from their somatic counterparts. In this review the information available on the common and distinct mechanisms of control of genome integrity utilized by different cell types along the self-renewal/differentiation program will be reviewed, with special emphasis on their roles in the prevention of aging and disease.

The Crosstalk between Gut Microbiota and White Adipose Tissue Mitochondria in Obesity.

Adipose tissue (AT) dysregulation is a key process in the pathophysiology of obesity and its cardiometabolic complications, but even if a growing body of evidence has been collected over recent decades, the underlying molecular basis of adiposopathy remains to be fully understood. In this context, mitochondria, the intracellular organelles that orchestrate energy production and undergo highly dynamic adaptive changes in response to changing environments, have emerged as crucial regulators of both white (WAT) and brown adipose tissue (BAT) metabolism and function. Given that the gut microbiota and its metabolites are able to regulate host metabolism, adipogenesis, WAT inflammation, and thermogenesis, we hypothesize that their frequently observed dysregulation in obesity could affect AT metabolism by exerting direct and indirect effects on AT mitochondria. By collecting and revising the current evidence on the connections between gut microbiota and AT mitochondria in obesity, we gained insights into the molecular biology of their hitherto largely unexplored crosstalk, tracing how gut microbiota may regulate AT mitochondrial function.

Trying to Understand the Complicated Taxonomy in Amaranthus (Amaranthaceae): Insights on Seeds Micromorphology.

Amaranthus is a genus taxonomically complex because of its high morphological variability, which led to nomenclatural disorders, misapplication of names, and misidentifications. Floristic and taxonomic studies on this genus are still incomplete, and many questions remain open. Seed micromorphology has been shown to play an important role in the taxonomy of plants. Regarding Amaranthaceae and Amaranthus, investigations are rare, and they refer to one or a few species. With the primary aim to test if seed features are helpful in the taxonomy of Amaranthus, we here present a detailed SEM study on seed micromorphology in 25 Amaranthus taxa using morphometric methods. Seeds were collected from field surveys and herbarium specimens; 14 seed coat features (7 qualitative and 7 quantitative) were measured on 111 samples (up to 5 seeds per sample). The results obtained revealed that seeds micromorphology provides interesting new taxonomic data concerning some taxa (species and below ranks). In fact, we were able to distinguish a few seed types, including one or more taxa, i.e., blitum-type, crassipes-type, deflexus-type, tuberculatus-type, and viridis-type. On the other hand, seed features are not useful for other species, for example, those included in the deflexus-type (A. deflexus, A. vulgatissimus, A. cacciatoi, A. spinosus, A. dubius, and A. stadleyanus). A diagnostic key of the studied taxa is proposed. Subgenera cannot be distinguished using seed features, thus confirming the published molecular data. All these facts reveal, once again, the taxonomic complexity of the genus Amaranthus since, e.g., just a few seed types can be defined.

Diet-related telomere shortening and chromosome stability.

Recent evidences have highlighted an influence of micronutrients in the maintenance of telomere length (TL). In order to explore whether diet-related telomere shortening had any physiological relevance and was accompanied by significant damage in the genome, in the present study, TL was assessed by terminal restriction fragment (TRF) analysis in peripheral blood lymphocytes of 56 healthy subjects for which detailed information on dietary habits was available and data were compared \with the incidence of nucleoplasmic bridges (NPBs), a marker of chromosomal instability related to telomere dysfunction visualised with the cytokinesis-blocked micronucleus assay. To increase the capability to detect even slight impairment of telomere function, the incidence of NPBs was also evaluated on cells exposed in vitro to ionising radiation. Care was taken to control for potential confounding factors that might influence TL, viz. age, hTERT genotype and smoking status. Data showed that higher consumption of vegetables was related with significantly higher mean TL (P = 0.013); in particular, the analysis of the association between micronutrients and mean TL highlighted a significant role of antioxidant intake, especially beta-carotene, on telomere maintenance (P = 0.004). However, the diet-related telomere shortening did not result in associated increased spontaneous or radiation-induced NPBs. The distribution of TRFs was also analysed and a slight prevalence of radiation-induced NPBs (P = 0.03) was observed in subjects with higher amount of very short TRFs (<2 kb). The relative incidence of very short TRFs was positively associate with ageing (P = 0.008) but unrelated to vegetables consumption and daily intake of micronutrients, suggesting that the degree of telomere erosion related with low dietary intake of antioxidants observed in this study was not so extensive to lead to chromosome instability.

Bariatric surgery-induced weight loss and associated genome-wide DNA-methylation alterations in obese individuals.

BACKGROUND: Obesity is a multifactorial and chronic condition of growing universal concern. It has recently been reported that bariatric surgery is a more successful treatment for severe obesity than other noninvasive interventions, resulting in rapid significant weight loss and associated chronic disease remission. The identification of distinct epigenetic patterns in patients who are obese or have metabolic imbalances has suggested a potential role for epigenetic alterations in causal or mediating pathways in the development of obesity-related pathologies. Specific changes in the epigenome (DNA methylome), associated with metabolic disorders, can be detected in the blood. We investigated whether such epigenetic changes are reversible after weight loss using genome-wide DNA methylome analysis of blood samples from individuals with severe obesity (mean BMI ~ 45) undergoing bariatric surgery. RESULTS: Our analysis revealed 41 significant (Bonferroni p < 0.05) and 1169 (false discovery rate p < 0.05) suggestive differentially methylated positions (DMPs) associated with weight loss due to bariatric surgery. Among the 41 significant DMPs, 5 CpGs were replicated in an independent cohort of BMI-discordant monozygotic twins (the heavier twin underwent diet-induced weight loss). The effect sizes of these 5 CpGs were consistent across discovery and replication sets (p < 0.05). We also identified 192 differentially methylated regions (DMRs) among which SMAD6 and PFKFB3 genes were the top hypermethylated and hypomethylated regions, respectively. Pathway enrichment analysis of the DMR-associated genes showed that functional pathways related to immune function and type 1 diabetes were significant. Weight loss due to bariatric surgery also significantly decelerated epigenetic age 12 months after the intervention (mean = - 4.29; p = 0.02). CONCLUSIONS: We identified weight loss-associated DNA-methylation alterations targeting immune and inflammatory gene pathways in blood samples from bariatric-surgery patients. The top hits were replicated in samples from an independent cohort of BMI-discordant monozygotic twins following a hypocaloric diet. Energy restriction and bariatric surgery thus share CpGs that may represent early indicators of response to the metabolic effects of weight loss. The analysis of bariatric surgery-associated DMRs suggests that epigenetic regulation of genes involved in endothelial and adipose tissue function is key in the pathophysiology of obesity.

A multi-marker integrative analysis reveals benefits and risks of bariatric surgery.

Bariatric surgery (BS) is an effective intervention for severe obesity and associated comorbidities. Although several studies have addressed the clinical and metabolic effects of BS, an integrative analysis of the complex body response to surgery is still lacking. We conducted a longitudinal data study with 36 patients with severe obesity who were tested before, 6 and 12 months after restrictive BS for more than one hundred blood biomarkers, including clinical, oxidative stress and metabolic markers, peptide mediators and red blood cell membrane lipids. By using a synthetic data-driven modeling based on principal component and correlation analyses, we provided evidence that, besides the early, well-known glucose metabolism- and weight loss-associated beneficial effects of BS, a tardive, weight-independent increase of the hepatic cholesterol metabolism occurs that is associated with potentially detrimental inflammatory and metabolic effects. Canonical correlation analysis indicated that oxidative stress is the most predictive feature of the BS-induced changes of both glucose and lipids metabolism. Our results show the power of multi-level correlation analysis to uncover the network of biological pathways affected by BS. This approach highlighted potential health risks of restrictive BS that are disregarded with the current practice to use weight loss as surrogate of BS success.

Increased levels of 5',8-Cyclopurine DNA lesions in inflammatory bowel diseases.

Chronic inflammation is estimated to be a causative factor in a variety of diseases. Under inflammatory conditions reactive oxygen species (ROS) and nitrogen species (RNS) are released leading to DNA damage accumulation and genomic instability. Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are oxidative DNA lesions, exclusively derived from the attack of HO• radicals, which are known to have cytotoxic and mutagenic properties. Herein, we have analyzed the presence of cPu in genomic DNA isolated from fresh colon and visceral adipose tissue biopsies collected from inflammatory bowel diseases (IBD)-affected patients and severely obese subjects, respectively, versus what observed in the control specimens. In colon biopsies, characterized by a higher gene expression level of inducible nitric oxide synthase (iNOS), a significant increase of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) lesions and an accumulation of both diastereomeric cPu have been detected. In contrast, the 8-oxo-dA and 8-oxo-dG levels were extremely lower compared to the colon tissues values and no accumulation of cPu, in the inflamed visceral adipose tissue biopsies isolated from bariatric patients versus the lean counterpart was reported. In addition, in adipose tissue undetectable levels of iNOS have been found. These data suggest a potential involvement of cPu in the colon cancer susceptibility observed in IBD patients.

Base damage and single-strand break repair: mechanisms and functional significance of short- and long-patch repair subpathways.

A large variety of DNA lesions induced by environmental agents or arising as an outcome of cellular metabolism are counteracted by a complex network of proteins that belong to the base excision repair/single strand break repair (BER/SSBR) processes. No matter whether the initial lesions are modified DNA bases or single-strand breaks with non-conventional termini these processes are completed either by replacement of a single (short-patch, SP) or more (long-patch, LP) nucleotides by different arrays of proteins. Here, the factors that are involved in the selection between SP- and LP-BER/SSBR are reviewed. The biological significance of these alternative subpathways is also presented as inferred from mutant mouse/cell models.

Hydrogen Peroxide-Induced DNA Damage and Repair through the Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

Human adipose-derived mesenchymal stem cells (hADMSCs) are recognized as a potential tool in cell tissue therapy because of their capacity to proliferate and differentiate in vitro. Several studies have addressed their use in regenerative medicine; however, little is known regarding their response to DNA damage and in particular to the reactive oxygen species (ROS) that are present in the microenvironment of implantation. In this study, we used the ROS-inducing agent hydrogen peroxide to explore the responses of (1) hADMSCs and (2) derived terminally differentiated adipocytes to oxidatively generated DNA damage. Using single cell gel electrophoresis, a dose-related increase was found for both DNA breaks and oxidative lesions (formamidopyrimidine DNA glycosylase-sensitive sites) upon exposure of hADMSCs to hydrogen peroxide. DNA repair capacity of hADMSCs was affected in cells exposed to 150 and 200 µM of hydrogen peroxide. An increase in the basal levels of DNA breaks and oxidative DNA lesions was observed through adipocyte differentiation. In addition, hydrogen peroxide-induced DNA damage increased through adipocyte differentiation; DNA repair capacity also decreased. This study is the first follow-up report on DNA repair capacity during adipogenic differentiation. Remarkably, in terminally differentiated adipocytes, DNA breakage repair is abolished while the repair of DNA oxidative lesions remains efficient.

The oxidized deoxynucleoside triphosphate pool is a significant contributor to genetic instability in mismatch repair-deficient cells.

Oxidation is a common form of DNA damage to which purines are particularly susceptible. We previously reported that oxidized dGTP is potentially an important source of DNA 8-oxodGMP in mammalian cells and that the incorporated lesions are removed by DNA mismatch repair (MMR). MMR deficiency is associated with a mutator phenotype and widespread microsatellite instability (MSI). Here, we identify oxidized deoxynucleoside triphosphates (dNTPs) as an important cofactor in this genetic instability. The high spontaneous hprt mutation rate of MMR-defective msh2(-/-) mouse embryonic fibroblasts was attenuated by expression of the hMTH1 protein, which degrades oxidized purine dNTPs. A high level of hMTH1 abolished their mutator phenotype and restored the hprt mutation rate to normal. Molecular analysis of hprt mutants showed that the presence of hMTH1 reduced the incidence of mutations in all classes, including frameshifts, and also implicated incorporated 2-oxodAMP in the mutator phenotype. In hMSH6-deficient DLD-1 human colorectal carcinoma cells, overexpression of hMTH1 markedly attenuated the spontaneous mutation rate and reduced MSI. It also reduced the incidence of -G and -A frameshifts in the hMLH1-defective DU145 human prostatic cancer cell line. Our findings indicate that incorporation of oxidized purines from the dNTP pool may contribute significantly to the extreme genetic instability of MMR-defective human tumors.

Base excision repair intermediates are mutagenic in mammalian cells.

Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5'-deoxyribose-5-phosphate (5'dRp) and 3'-[2,3-didehydro-2,3-dideoxy-ribose] (3'ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the 'A-rule' and is also consistent with the use of the 5' neighbouring base to instruct nucleotide incorporation (5'-rule). Preferential adenine insertion was also observed after in vivo replication of 5'dRp or 3'ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives 'faceless' BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations.

Single nucleotide polymorphisms in DNA glycosylases: From function to disease.

Oxidative stress is associated with a growing number of diseases that span from cancer to neurodegeneration. Most oxidatively induced DNA base lesions are repaired by the base excision repair (BER) pathway which involves the action of various DNA glycosylases. There are numerous genome wide studies attempting to associate single-nucleotide polymorphisms (SNPs) with predispositions to various types of disease; often, these common variants do not have significant alterations in their biochemical function and do not exhibit a convincing phenotype. Nevertheless several lines of evidence indicate that SNPs in DNA repair genes may modulate DNA repair capacity and contribute to risk of disease. This overview provides a convincing picture that SNPs of DNA glycosylases that remove oxidatively generated DNA lesions are susceptibility factors for a wide disease spectrum that includes besides cancer (particularly lung, breast and gastrointestinal tract), cochlear/ocular disorders, myocardial infarction and neurodegenerative disorders which can be all grouped under the umbrella of oxidative stress-related pathologies.

Crosstalk between mismatch repair and base excision repair in human gastric cancer.

DNA repair gene expression in a set of gastric cancers suggested an inverse association between the expression of the mismatch repair (MMR) gene MLH1 and that of the base excision repair (BER) gene DNA polymerase ß (Polß). To gain insight into possible crosstalk of these two repair pathways in cancer, we analysed human gastric adenocarcinoma AGS cells over-expressing Polß or Polß active site mutants, alone or in combination with MLH1 silencing. Next, we investigated the cellular response to the alkylating agent methyl methanesulfonate (MMS) and the purine analogue 6-thioguanine (6-TG), agents that induce lesions that are substrates for BER and/or MMR. AGS cells over-expressing Polß were resistant to 6-TG to a similar extent as when MLH1 was inactivated while inhibition of O6-methylguanine-DNA methyltransferase (MGMT) was required to detect resistance to MMS. Upon either treatment, the association with MLH1 down-regulation further amplified the resistant phenotype. Moreover, AGS cells mutated in Polß were hypersensitive to both 6-TG and MMS killing and their sensitivity was partially rescued by MLH1 silencing. We provide evidence that the critical lethal lesions in this new pathway are double strand breaks that are exacerbated when Polß is defective and relieved when MLH1 is silenced. In conclusion, we provide evidence of crosstalk between MLH1 and Polß that modulates the response to alkylation damage. These studies suggest that the Polß/MLH1 status should be taken into consideration when designing chemotherapeutic approaches for gastric cancer.

Overexpression of parkin rescues the defective mitochondrial phenotype and the increased apoptosis of Cockayne Syndrome A cells.

The ERCC8/CSA gene encodes a WD-40 repeat protein (CSA) that is part of a E3-ubiquitin ligase/COP9 signalosome complex. When mutated, CSA causes the Cockayne Syndrome group A (CS-A), a rare recessive progeroid disorder characterized by sun sensitivity and neurodevelopmental abnormalities. CS-A cells features include ROS hyperproduction, accumulation of oxidative genome damage, mitochondrial dysfunction and increased apoptosis that may contribute to the neurodegenerative process. In this study, we show that CSA localizes to mitochondria and specifically interacts with the mitochondrial fission protein dynamin-related protein (DRP1) that is hyperactivated when CSA is defective. Increased fission is not counterbalanced by increased mitophagy in CS-A cells thus leading to accumulation of fragmented mitochondria. However, when mitochondria are challenged with the mitochondrial toxin carbonyl cyanide m-chloro phenyl hydrazine, CS-A fibroblasts undergo mitophagy as efficiently as normal fibroblasts, suggesting that this process remains targetable to get rid of damaged mitochondria. Indeed, when basal mitophagy was potentiated by overexpressing Parkin in CSA deficient cells, a significant rescue of the dysfunctional mitochondrial phenotype was observed. Importantly, Parkin overexpression not only reactivates basal mitophagy, but plays also an anti-apoptotic role by significantly reducing the translocation of Bax at mitochondria in CS-A cells. These findings provide new mechanistic insights into the role of CSA in mitochondrial maintenance and might open new perspectives for therapeutic approaches.

In vitro base excision repair assay using mammalian cell extracts.

Base excision repair (BER) is the main pathway for removal of endogenous DNA damage. This repair mechanism is initiated by a specific DNA glycosylase that recognizes and removes the damaged base through N-glycosylic bond hydrolysis. The generated apurinic/apyrimidinic (AP) site can be repaired in mammalian cells by two alternative pathways which involve either the replacement of one (short patch BER) or more nucleotides (long patch BER) at the lesion site. This chapter describes a repair replication assay for measuring BER efficiency and mode in mammalian cell extracts. The DNA substrate used in the assay is either a randomly depurinated plasmid DNA or a plasmid containing a single lesion that is processed via BER (for example a single AP site or uracil residue). The construction of a single lesion at a defined site of the plasmid genome makes the substrate amenable to fine mapping of the repair patches, thus allowing discrimination between the two BER pathways.