Klotho gene improves oxidative stress injury after myocardial infarction.

The aim of the present study was to investigate the effects and mechanisms of the Klotho gene in oxidative stress injury after myocardial infarction. Sprague-Dawley rats were divided into five groups (sham, model, pDC316, LY294002, and pDC316-Klotho). Subsequently, the superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) concentrations were measured in myocardial tissues. Additionally, pathological differences among the groups were evaluated using hematoxylin and eosin and Masson's trichrome staining. Apoptosis was assayed by terminal deoxynucleotidyl transferase 2'-deoxyuridine-5'-triphosphate nick end-labeling assay, evaluated Klotho protein expression by immunohistochemical assay, and assessed Nrf 2 and ARE protein expressions using western blotting assay. As compared with in the sham group, the SOD, MDA, and GSH concentrations were significantly deteriorated (P<0.001, respectively); cardiomyocyte apoptosis index values were significantly increased (P<0.001); Klotho protein expression was significantly depressed; and Nrf-2 and ARE protein expressions were significantly (P<0.001, respectively) in the model and pDC316 groups. However, with Klotho supplementation by pDC316 transfection, as compared with in the model group, the SOD, MDA, and GSH concentrations were significantly improved (P<0.001, respectively); the cardiomyocyte apoptosis index values were significantly suppressed (P<0.001); and the pathology was improved. Further, the Klotho protein expression of the pDC316-Klotho group was significantly upregulated and the Nrf-2 and ARE proteins expressions of the LY294002 and pDC316-Klotho groups were significantly suppressed. Klotho overexpression improved findings of oxidative stress injury after myocardial infarction.

Efficacy and safety of bempedoic acid alone or combining with other lipid-lowering therapies in hypercholesterolemic patients: a meta-analysis of randomized controlled trials.

BACKGROUND: Bempedoic acid is a new drug that reduces cholesterol synthesis via inhibiting ATP citrate lyase. It remains unclear whether the combination of bempedoic acid and other lipid-lowering drugs is better than these drugs alone. This study systematically reviewed the efficacy and safety of bempedoic acid monotherapy or combination togethers in hypercholesterolemic patients. METHODS: Randomized controlled trials were searched across Medline, Embase, Cochrane library, web of science, etc. The net change scores [least squares mean (LSM) percentage change] in LDL-C level were meta-analyzed using weighted mean difference. The reductions in other lipids including total cholesterol (TC), non-high-density lipoprotein cholesterol (non-HDL-C) and apolipoprotein (ApoB) and high sensitivity C reactive protein (hsCRP) were also assessed. Odds ratio (OR) of the incidence of adverse events (AEs) were calculated to evaluate the safety of bempedoic acid. RESULTS: A total of 13 trials (4858 participates) were included. Pooled data showed that the combination togethers resulted in greater reductions in LDL-C level than monotherapies (bempedoic acid + statin vs. statin: LSM difference (%), - 18.37, 95% CI, - 20.16 to - 16.57, I2 = 0; bempedoic acid + ezetimibe vs. ezetimibe: LSM difference (%), - 18.89, 95% CI, - 29.66 to - 8.13, I2 = 87%). But the difference in efficacy between bempedoic acid and ezetimibe was not obvious. Meta-regression analysis showed the treatment duration was a source of heterogeneity (adj R2 = 16.92, 95% CI, 0.04 to 0.72). Furthermore, the background therapy of statin before screening decreased the efficacy of bempedoic acid. In addition, bempedoic acid also resulted in a significant reduction in TC, non-HDL-C, ApoB and hsCRP level. The OR of muscle-related AEs by the combination of bempedoic acid and statin was 1.29 (95% CI, 1.00 to 1.67, I2 = 0) when compared with statin alone. CONCLUSION: This study showed the efficacy of combination togethers were similar but stronger than these drugs alone. Of note, a trend of high risk of muscle-related AEs by the combination of bempedoic acid and statin was observed, though it is not statistically significant, such risk is needed to be confirmed by more trials, because it is important for us to determine which is the better combinative administration for statin-intolerant patients.

Response to Comment on "DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification".

Following the Comment of Stewart et al, we repeated our analysis on sequencing runs from The Cancer Genome Atlas (TCGA) using their suggested parameters. We found signs of oxidative damage in all sequence contexts and irrespective of the sequencing date, reaffirming that DNA damage affects mutation-calling pipelines in their ability to accurately identify somatic variations.

DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification.

Mutations in somatic cells generate a heterogeneous genomic population and may result in serious medical conditions. Although cancer is typically associated with somatic variations, advances in DNA sequencing indicate that cell-specific variants affect a number of phenotypes and pathologies. Here, we show that mutagenic damage accounts for the majority of the erroneous identification of variants with low to moderate (1 to 5%) frequency. More important, we found signatures of damage in most sequencing data sets in widely used resources, including the 1000 Genomes Project and The Cancer Genome Atlas, establishing damage as a pervasive cause of sequencing errors. The extent of this damage directly confounds the determination of somatic variants in these data sets.

Differential strand separation at critical temperature: a minimally disruptive enrichment method for low-abundance unknown DNA mutations.

Detection of low-level DNA variations in the presence of wild-type DNA is important in several fields of medicine, including cancer, prenatal diagnosis and infectious diseases. PCR-based methods to enrich mutations during amplification have limited multiplexing capability, are mostly restricted to known mutations and are prone to polymerase or mis-priming errors. Here, we present Differential Strand Separation at Critical Temperature (DISSECT), a method that enriches unknown mutations of targeted DNA sequences purely based on thermal denaturation of DNA heteroduplexes without the need for enzymatic reactions. Target DNA is pre-amplified in a multiplex reaction and hybridized onto complementary probes immobilized on magnetic beads that correspond to wild-type DNA sequences. Presence of any mutation on the target DNA forms heteroduplexes that are subsequently denatured from the beads at a critical temperature and selectively separated from wild-type DNA. We demonstrate multiplexed enrichment by 100- to 400-fold for KRAS and TP53 mutations at multiple positions of the targeted sequence using two to four successive cycles of DISSECT. Cancer and plasma-circulating DNA samples containing traces of mutations undergo mutation enrichment allowing detection via Sanger sequencing or high-resolution melting. The simplicity, scalability and reliability of DISSECT make it a powerful method for mutation enrichment that integrates well with existing downstream detection methods.

Temperature-tolerant COLD-PCR reduces temperature stringency and enables robust mutation enrichment.

BACKGROUND: Low-level mutations in clinical tumor samples often reside below mutation detection limits, thus leading to false negatives that may impact clinical diagnosis and patient management. COLD-PCR (coamplification at lower denaturation temperature PCR) is a technology that magnifies unknown mutations during PCR, thus enabling downstream mutation detection. However, a practical difficulty in applying COLD-PCR has been the requirement for strict control of the denaturation temperature for a given sequence, to within ±0.3 °C. This requirement precludes simultaneous mutation enrichment in sequences of substantially different melting temperature (T(m)) and limits the technique to a single sequence at a time. We present a temperature-tolerant (TT) approach (TT-COLD-PCR) that reduces this obstacle. METHODS: We describe thermocycling programs featuring a gradual increase of the denaturation temperature during COLD-PCR. This approach enabled enrichment of mutations when the cycling achieves the appropriate critical denaturation temperature of each DNA amplicon that is being amplified. Validation was provided for KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) and TP53 (tumor protein p53) exons 6-9 by use of dilutions of mutated DNA, clinical cancer samples, and plasma-circulating DNA. RESULTS: A single thermocycling program with a denaturation-temperature window of 2.5-3.0 °C enriches mutations in all DNA amplicons simultaneously, despite their different T(m)s. Mutation enrichments of 6-9-fold were obtained with TT-full-COLD-PCR. Higher mutation enrichments were obtained for the other 2 forms of COLD-PCR, fast-COLD-PCR, and ice-COLD-PCR. CONCLUSIONS: Low-level mutations in diverse amplicons with different T(m)s can be mutation enriched via TT-COLD-PCR provided that their T(m)s fall within the denaturation-temperature window applied during amplification. This approach enables simultaneous enrichment of mutations in several amplicons and increases significantly the versatility of COLD-PCR.

Rapid and sensitive detection of KRAS mutation after fast-COLD-PCR enrichment and high-resolution melting analysis.

KRAS mutations exhibit significant predictive and prognostic value in cancer. Efficient, sensitive, and accurate molecular approaches are required to evaluate KRAS mutation status, even when mutant alleles are restricted to a small portion of a clinical sample, which otherwise contains wild-type alleles. We describe a highly sensitive method to detect KRAS mutations by high-resolution melting (HRM) analysis after mutation enrichment by fast-COLDpolymerase chain reaction (PCR). Using 10 ng of starting DNA and after fast-COLD-PCR of a 76-bp region containing KRAS codons 12 and 13; the amplicons undergo a nested conventional PCR reaction followed by HRM analysis. Samples exhibiting aberrant melting profiles are sequenced to identify mutation type and position. Serial dilution experiments indicate a sensitivity of approximately 0.3% mutant-to-wild type for HRM-based mutation detection and the ability to directly sequence mutation-containing samples. A number of lung adenocarcinoma specimens earlier characterized were screened. Fast-COLD-PCR-HRM analysis correctly identified KRAS mutations and also showed a previously undetected, low-level missense GGT > TTT complex mutation. On account of the short target regions and low requirement of starting DNA, this rapid, cost-effective, and sensitive fast-COLD-PCR-HRM approach is expected to find broad application for detecting low-abundance KRAS mutations in a wide range of clinical specimens.

Multiplex amplification coupled with COLD-PCR and high resolution melting enables identification of low-abundance mutations in cancer samples with low DNA content.

Thorough screening of cancer-specific biomarkers, such as DNA mutations, can require large amounts of genomic material; however, the amount of genomic material obtained from some specimens (such as biopsies, fine-needle aspirations, circulating-DNA or tumor cells, and histological slides) may limit the analyses that can be performed. Furthermore, mutant alleles may be at low-abundance relative to wild-type DNA, reducing detection ability. We present a multiplex-PCR approach tailored to amplify targets of interest from small amounts of precious specimens, for extensive downstream detection of low-abundance alleles. Using 3 ng of DNA (1000 genome-equivalents), we amplified the 1 coding exons (2-11) of TP53 via multiplex-PCR. Following multiplex-PCR, we performed COLD-PCR (co-amplification of major and minor alleles at lower denaturation temperature) to enrich low-abundance variants and high resolution melting (HRM) to screen for aberrant melting profiles. Mutation-positive samples were sequenced. Evaluation of mutation-containing dilutions revealed improved sensitivities after COLD-PCR over conventional-PCR. COLD-PCR improved HRM sensitivity by approximately threefold to sixfold. Similarly, COLD-PCR improved mutation identification in sequence-chromatograms over conventional PCR. In clinical specimens, eight mutations were detected via conventional-PCR-HRM, whereas 12 were detected by COLD-PCR-HRM, yielding a 33% improvement in mutation detection. In summary, we demonstrate an efficient approach to increase screening capabilities from limited DNA material via multiplex-PCR and improve mutation detection sensitivity via COLD-PCR amplification.

COLD-PCR: improving the sensitivity of molecular diagnostics assays.

The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics, individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance variants is a pronounced limitation of most currently available molecular assays. We have recently developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation. This novel form of PCR selectively amplifies low-abundance DNA variants from mixtures of wild-type and mutant-containing (or variant-containing) sequences, irrespective of the mutation type or position on the amplicon, by using a critical denaturation temperature. The use of a lower denaturation temperature in COLD-PCR results in selective denaturation of amplicons with mutation-containing molecules within wild-type mutant heteroduplexes or with a lower melting temperature. COLD-PCR can be used in lieu of conventional PCR in several molecular applications, thus enriching the mutant fraction and improving the sensitivity of downstream mutation detection by up to 100-fold.

DNA repair in mammalian mitochondria: Much more than we thought?

For many years, the repair of most damage in mitochondrial DNA (mtDNA) was thought limited to short-patch base excision repair (SP-BER), which replaces a single nucleotide by the sequential action of DNA glycosylases, an apurinic/apyrimidinic (AP) endonuclease, the mitochondrial DNA polymerase gamma, an abasic lyase activity, and mitochondrial DNA ligase. However, the likely array of lesions inflicted on mtDNA by oxygen radicals and the possibility of replication errors and disruptions indicated that such a restricted repair repertoire would be inadequate. Recent studies have considerably expanded our knowledge of mtDNA repair to include long-patch base excision repair (LP-BER), mismatch repair, and homologous recombination and nonhomologous end-joining. In addition, elimination of mutagenic 8-oxodeoxyguanosine triphosphate (8-oxodGTP) helps prevent cell death due to the accumulation of this oxidation product in mtDNA. Although it was suspected for many years that irreparably damaged mtDNA might be targeted for degradation, only recently was clear evidence provided for this hypothesis. Therefore, multiple DNA repair pathways and controlled degradation of mtDNA function together to maintain the integrity of mitochondrial genome.

Removal of oxidative DNA damage via FEN1-dependent long-patch base excision repair in human cell mitochondria.

Repair of oxidative DNA damage in mitochondria was thought limited to short-patch base excision repair (SP-BER) replacing a single nucleotide. However, certain oxidative lesions cannot be processed by SP-BER. Here we report that 2-deoxyribonolactone (dL), a major type of oxidized abasic site, inhibits replication by mitochondrial DNA (mtDNA) polymerase gamma and interferes with SP-BER by covalently trapping polymerase gamma during attempted dL excision. However, repair of dL was detected in human mitochondrial extracts, and we show that this repair is via long-patch BER (LP-BER) dependent on flap endonuclease 1 (FEN1), not previously known to be present in mitochondria. FEN1 was retained in protease-treated mitochondria and detected in mitochondrial nucleoids that contain known mitochondrial replication and transcription proteins. Results of immunofluorescence and subcellular fractionation studies were also consistent with the presence of FEN1 in the mitochondria of intact cells. Immunodepletion experiments showed that the LP-BER activity of mitochondrial extracts was strongly diminished in parallel with the removal of FEN1, although some activity remained, suggesting the presence of an additional flap-removing enzyme. Biological evidence for a FEN1 role in repairing mitochondrial oxidative DNA damage was provided by RNA interference experiments, with the extent of damage greater and the recovery slower in FEN1-depleted cells than in control cells. The mitochondrial LP-BER pathway likely plays important roles in repairing dL lesions and other oxidative lesions and perhaps in normal mtDNA replication.

Mechanisms of base selection by the Escherichia coli mispaired uracil glycosylase.

The repair of the multitude of single-base lesions formed daily in cells of all living organisms is accomplished primarily by the base excision repair pathway that initiates repair through a series of lesion-selective glycosylases. In this article, single-turnover kinetics have been measured on a series of oligonucleotide substrates containing both uracil and purine analogs for the Escherichia coli mispaired uracil glycosylase (MUG). The relative rates of glycosylase cleavage have been correlated with the free energy of helix formation and with the size and electronic inductive properties of a series of uracil 5-substituents. Data are presented that MUG can exploit the reduced thermodynamic stability of mispairs to distinguish U:A from U:G pairs. Discrimination against the removal of thymine results primarily from the electron-donating property of the thymine 5-methyl substituent, whereas the size of the methyl group relative to a hydrogen atom is a secondary factor. A series of parameters have been obtained that allow prediction of relative MUG cleavage rates that correlate well with observed relative rates that vary over 5 orders of magnitude for the series of base analogs examined. We propose that these parameters may be common among DNA glycosylases; however, specific glycosylases may focus more or less on each of the parameters identified. The presence of a series of glycosylases that focus on different lesion properties, all coexisting within the same cell, would provide a robust and partially redundant repair system necessary for the maintenance of the genome.

Terminally differentiated muscle cells are defective in base excision DNA repair and hypersensitive to oxygen injury.

The differentiation of skeletal myoblasts is characterized by permanent withdrawal from the cell cycle and fusion into multinucleated myotubes. Muscle cell survival is critically dependent on the ability of cells to respond to oxidative stress. Base excision repair (BER) is the main repair mechanism of oxidative DNA damage. In this study, we compared the levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8-hydroxyguanine DNA levels in terminally differentiated cells, suggested that reactive oxygen species are produced during this process. The repair of 2-deoxyribonolactone, which is exclusively processed by long-patch BER, was impaired in cell extracts from myotubes. The repair of a natural abasic site (a preferred substrate for short-patch BER) also was delayed. The defect in BER of terminally differentiated muscle cells was ascribed to the nearly complete lack of DNA ligase I and to the strong down-regulation of XRCC1 with subsequent destabilization of DNA ligase IIIalpha. The attenuation of BER in myotubes was associated with significant accumulation of DNA damage as detected by increased DNA single-strand breaks and phosphorylated H2AX nuclear foci upon exposure to hydrogen peroxide. We propose that in skeletal muscle exacerbated by free radical injury, the accumulation of DNA repair intermediates, due to attenuated BER, might contribute to myofiber degeneration as seen in sarcopenia and many muscle disorders.

ATF4-dependent oxidative induction of the DNA repair enzyme Ape1 counteracts arsenite cytotoxicity and suppresses arsenite-mediated mutagenesis.

Arsenite is a human carcinogen causing skin, bladder, and lung tumors, but the cellular mechanisms underlying these effects remain unclear. We investigated expression of the essential base excision DNA repair enzyme apurinic endonuclease 1 (Ape1) in response to sodium arsenite. In mouse 10T(1/2) fibroblasts, Ape1 induction in response to arsenite occurred about equally at the mRNA, protein, and enzyme activity levels. Analysis of the APE1 promoter region revealed an AP-1/CREB binding site essential for arsenite-induced transcriptional activation in both mouse and human cells. Electrophoretic mobility shift assays indicated that an ATF4/c-Jun heterodimer was the responsible transcription factor. RNA interference targeting c-Jun or ATF4 eliminated arsenite-induced APE1 transcription. Suppression of Ape1 or ATF4 sensitized both mouse fibroblasts (10T(1/2)) and human lymphoblastoid cells (TK6) to arsenite cytotoxicity. Expression of Ape1 from a transgene did not efficiently restore arsenite resistance in ATF4-depleted cells but did offset initial accumulation of abasic DNA damage following arsenite treatment. Mutagenesis by arsenite (at the TK and HPRT loci in TK6 cells) was observed only for ATF4-depleted cells, which was strongly offset by Ape1 expression from a transgene. Therefore, the ATF4-mediated up-regulation of Ape1 and other genes plays a key role against arsenite-mediated toxicity and mutagenesis.

Impact of cytosine 5-halogens on the interaction of DNA with restriction endonucleases and methyltransferase.

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.

Genomic instability and aging-like phenotype in the absence of mammalian SIRT6.

The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.

5-halogenated pyrimidine lesions within a CpG sequence context mimic 5-methylcytosine by enhancing the binding of the methyl-CpG-binding domain of methyl-CpG-binding protein 2 (MeCP2).

Perturbations in cytosine methylation signals are observed in the majority of human tumors; however, it is as yet unknown how methylation patterns become altered. Epigenetic changes can result in the activation of transforming genes as well as in the silencing of tumor suppressor genes. We report that methyl-CpG-binding proteins (MBPs), specific for methyl-CpG dinucleotides, bind with high affinity to halogenated pyrimidine lesions, previously shown to result from peroxidase-mediated inflammatory processes. Emerging data suggest that the initial binding of MBPs to methyl-CpG sequences may be a seeding event that recruits chromatin-modifying enzymes and DNA methyltransferase, initiating a cascade of events that result in gene silencing. MBD4, a protein with both methyl-binding and glycosylase activity demonstrated repair activity against a series of 5-substituted pyrimidines, with the greatest efficiency against 5-chlorouracil, but undetectable activity against 5-chlorocytosine. The data presented here suggest that halogenated pyrimidine damage products can potentially accumulate and mimic endogenous methylation signals.

Synthesis and characterization of oligonucleotides containing 5-chlorocytosine.

Recent studies have shown that reactive chlorine species, derived from myeloperoxidase-mediated inflammation responses, can modify DNA bases, generating 5-chloropyrimidines. The chlorinated adducts could be mutagenic or perturb DNA-protein interactions; however, the biological significance of these adducts is as yet unknown. We report here a method for the synthesis of 5-chlorocytosine- (ClC-) containing oligonucleotides that will be used in subsequent biochemical and biophysical studies to determine the consequences of pyrimidine chlorination. The ClC-phosphoramidite synthon is obtained by chlorination of 2'-deoxyuridine followed by conversion to the O(4)-ethyl analogue. The amino group needed to form the corresponding cytosine derivative is added by displacement of the O(4)-ethyl group during ammonia deprotection. A battery of methods, including mass spectrometry, has been used to characterize oligonucleotides containing ClC. Following oligonucleotide synthesis and deprotection, only trace amounts of the deamination product 5-chlorouracil can be detected by enzymatic cleavage of duplex oligonucleotides with the mispaired uracil glycosylase, MUG. In contrast to previous reports, we find that ClC is more stable in DNA than anticipated. Approximately 20% ClC is lost under standard formic acid hydrolysis conditions (88% formic acid, 140 degrees C, 30 min), while only 5% is recovered as 5-chlorouracil (ClU).

DNA ligases ensure fidelity by interrogating minor groove contacts.

DNA ligases, found in both prokaryotes and eukaryotes, covalently link the 3'-hydroxyl and 5'-phosphate ends of duplex DNA segments. This reaction represents a completion step for DNA replication, repair and recombination. It is well established that ligases are sensitive to mispairs present on the 3' side of the ligase junction, but tolerant of mispairs on the 5' side. While such discrimination would increase the overall accuracy of DNA replication and repair, the mechanisms by which this fidelity is accomplished are as yet unknown. In this paper, we present the results of experiments with Tth ligase from Thermus thermophilus HB8 and a series of nucleoside analogs in which the mechanism of discrimination has been probed. Using a series of purine analogs substituted in the 2 and 6 positions, we establish that the apparent base pair geometry is much more important than relative base pair stability and that major groove contacts are of little importance. This result is further confirmed using 5-fluorouracil (FU) mispaired with guanine. At neutral pH, the FU:G mispair on the 3' side of a ligase junction is predominantly in a neutral wobble configuration and is poorly ligated. Increasing the solution pH increases the proportion of an ionized base pair approximating Watson-Crick geometry, substantially increasing the relative ligation efficiency. These results suggest that the ligase could distinguish Watson-Crick from mispaired geometry by probing the hydrogen bond acceptors present in the minor groove as has been proposed for DNA polymerases. The significance of minor groove hydrogen bonding interactions is confirmed with both Tth and T4 DNA ligases upon examination of base pairs containing the pyrimidine shape analog, difluorotoluene (DFT). Although DFT paired with adenine approximates Watson-Crick geometry, a minor groove hydrogen bond acceptor is lost. Consistent with this hypothesis, we observe that DFT-containing base pairs inhibit ligation when on the 3' side of the ligase junction. The NAD+-dependent ligase, Tth, is more sensitive to the DFT analog on the unligated strand whereas the ATP-dependent T4 ligase is more sensitive to substitutions in the template strand. Electrophoretic gel mobility-shift assays demonstrate that the Tth ligase binds poorly to oligonucleotide substrates containing analogs with altered minor groove contacts.