Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.
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PMID:Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis. 1140 38

Knowledge of inherited and sporadic mutations in known and candidate cancer genes may influence clinical decisions. We have developed a mutation scanning method that combines thermostable EndonucleaseV (Endo V) and DNA ligase. Variant and wild-type PCR amplicons are generated using fluorescently labeled primers, and heteroduplexed. Thermotoga maritima (Tma) EndoV recognizes and primarily cleaves heteroduplex DNA one base 3' to the mismatch, as well as nicking matched DNA at low levels. Thermus species (Tsp.) AK16D DNA ligase reseals the background nicks to create a highly sensitive and specific assay. The fragment mobility on a DNA sequencing gel reveals the approximate position of the mutation. This method identified 31/35 and 8/8 unique point mutations and insertions/deletions, respectively, in the p53, VHL, K-ras, APC, BRCA1, and BRCA2 genes. The method has the sensitivity to detect K-ras mutations diluted 1 : 20 with wild-type DNA, a p53 mutation in a 1.7 kb amplicon, and unknown p53 mutations in pooled DNA samples. EndoV/Ligase mutation scanning combined with PCR/LDR/Universal array proved superior to automated DNA sequencing for detecting p53 mutations in colon tumors. This technique is well suited for scanning low-frequency mutations in pooled samples and for analysing tumor DNA containing a minority of the unknown mutation.
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PMID:An endonuclease/ligase based mutation scanning method especially suited for analysis of neoplastic tissue. 1189 24

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.
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PMID:Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach. 1213 76

O6-methylguanin-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups from O6-methylguanine to itself. Alkylation of DNA at the O6 position of guanine is the first step by alkylating agents in inducing DNA mutations in an organism. When MGMT and the mismatch repair (MMR) system are impaired, O6-methylguanine mispairs with thymine during DNA replication, resulting in a G:C right curved arrow A:T transitional mutation in DNA. We obtained cancer lesions by manual micro-dissection (MMD) from 26 paraffin-embedded formalin-fixed gallbladder carcinoma and Laser Capture Micro-dissection (LCM) method from 10 fresh frozen specimens. Mutation analysis was performed on the micro-dissected samples for K-ras and beta-catenin genes. At codon 12 of the K-ras gene, the MMD and LCM methods detected mutations in 3 (11.5%) and 1 (10%) case, respectively. In exon 3 of beta-catenin gene, only 1 (3.8%) case revealed a mutation in MMD cancer foci. Two cases without MGMT or MMR expression revealed a G right curved arrow A transition mutation in the K-ras gene. The findings suggested that negative MGMT and MMR status contributed to a G:C right curved arrow A:T transitional mutation in the K-ras gene. However, K-ras and beta-catenin mutations were actually rare in GB carcinoma. Other gene mutations frequently occurring in gallbladder carcinoma might be affected by this negative MGMT and MMR status.
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PMID:Mutation analysis of K-ras and beta-catenin genes related to O6-methylguanin-DNA methyltransferase and mismatch repair protein status in human gallbladder carcinoma. 1246 20

The ability to associate mutations in cancer genes with the disease and its subtypes is critical for understanding oncogenesis and identifying biomarkers for clinical diagnosis. A two-step mutation scanning method that sequentially used endonuclease V (EndoV) to nick at mismatches and DNA ligase to reseal incorrectly or nonspecifically nicked sites was previously developed in our laboratory. Herein we report an optimized single-step assay that enables ligase to proofread EndoV cleavage in real-time under a compromise between buffer conditions. Real-time proofreading results in a dramatic reduction of background cleavage. A universal PCR strategy that employs both unlabeled gene-specific primers and labeled universal primers, allows for multiplexed gene amplification and precludes amplification of primer dimers. Internally labeled PCR primers eliminate EndoV cleavage at the 5' terminus, enabling high-throughput capillary electrophoresis readout. Furthermore, signal intensity is increased and artifacts are reduced by generating heteroduplexes containing only one of the two possible mismatches (e.g. either A/C or G/T). The single-step assay improves sensitivity to 1:50 and 1:100 (mutant:wild type) for unknown mutations in the p53 and K-ras genes, respectively, opening prospects as an early detection tool.
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PMID:High sensitivity EndoV mutation scanning through real-time ligase proofreading. 1551 9

The present study reported proof-of-principle for a genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) through the gold nanoparticle assembly and the ligase reaction. By incorporating the high-fidelity DNA ligase (Tth DNA ligase) into the allele-specific ligation-based gold nanoparticle assembly, this assay provided a convenient yet powerful colorimetric detection that enabled a straightforward single-base discrimination without the need of precise temperature control. Additionally, the ligase reaction can be performed at a relatively high temperature, which offers the benefit for mitigating the non-specific assembly of gold nanoparticles induced by interfering DNA strands. The assay could be implemented via three steps: a hybridization reaction that allowed two gold nanoparticle-tagged probes to hybrid with the target DNA strand, a ligase reaction that generates the ligation between perfectly matched probes while no ligation occurred between mismatched ones and a thermal treatment at a relatively high temperature that discriminate the ligation of probes. When the reaction mixture was heated to denature the formed duplex, the purple color of the perfect-match solution would not revert to red, while the mismatch gave a red color as the assembled gold nanoparticles disparted. The present approach has been demonstrated with the identification of a single-base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild-type and mutant type were successfully scored. To our knowledge, this was the first report concerning SNP detection based on the ligase reaction and the gold nanoparticle assembly. Owing to its ease of operation and high specificity, it was expected that the proposed procedure might hold great promise in practical clinical diagnosis of gene-mutant diseases.
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PMID:A colorimetric method for point mutation detection using high-fidelity DNA ligase. 1625 79

Endonuclease V (endo V) recognizes a broad range of aberrations in DNA such as deaminated bases or mismatches. It nicks DNA at the second phosphodiester bond 3' to a deaminated base or a mismatch. Endonuclease V obtained from Thermotoga maritima preferentially cleaves purine mismatches in certain sequence context. Endonuclease V has been combined with a high-fidelity DNA ligase to develop an enzymatic method for mutation scanning. A biochemical screening of site-directed mutants identified mutants in motifs III and IV that altered the base preferences in mismatch cleavage. Most profoundly, a single alanine substitution at Y80 position switched the enzyme to essentially a C-specific mismatch endonuclease, which recognized and cleaved A/C, C/A, T/C, C/T and even the previously refractory C/C mismatches. Y80A can also detect the G13D mutation in K-ras oncogene, an A/C mismatch embedded in a G/C rich sequence context that was previously inaccessible using the wild-type endo V. This investigation offers insights on base recognition and active site organization. Protein engineering in endo V may translate into better tools in mutation recognition and cancer mutation scanning.
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PMID:Switching base preferences of mismatch cleavage in endonuclease V: an improved method for scanning point mutations. 1713 Jan 53

The combination of suspension array with rolling circle amplification can lead to a sensitive and specific assay for single-nucleotide polymorphisms (SNPs) detection, as demonstrated in this study. A circular template generated by ligation upon the recognition of a point mutation on DNA targets was amplified isothermally by the Phi29 polymerase on microspheres. The elongation products were labeled with fluorochrome-tagged probes and detected in a flow cytometer, indicating the mutation occurrence. As low as 10 amol of mutated strands was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, which could be attributed to the high amplification efficiency of Phi29, the high binding capacity of the microspheres, and the remarkable precision of DNA ligase in distinguishing mismatched bases at the ligation site. A novel design of using two differently labeled detection probes on the same microsphere to target both the wild-type and mutant samples allowed parallel determination of the heterozygosity for two SNPs (K-ras G12C and TP53 R273H) in PCR amplicons prepared from human genomic DNA extracts. This ability lays the groundwork for further enhancing the assay throughput by using multiple fluorophores and microspheres with distinct properties.
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PMID:Typing of multiple single-nucleotide polymorphisms by a microsphere-based rolling circle amplification assay. 1797 2

A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5'-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90 degrees C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.
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PMID:Electrochemical detection of point mutation based on surface ligation reaction and biometallization. 1824 73