Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04637 (p53)
 77,613 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific carcinogen associated with lung cancer. Its complex enzymatic activation, leading to methyl and pyridyloxobutyl (POB)-modified DNA, makes DNA damage difficult to characterize and quantify. Therefore, we use the NNK analogue 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) to induce damage in genomic DNA, and to map the sites and frequency of adducts at nucleotide resolution using ligation-mediated polymerase chain reaction and terminal transferase-dependent polymerase chain reactions (LMPCR and TDPCR). NNKOAc induced single-strand breaks in a concentration-dependent manner. Post-alkylation treatments, including hot piperidine or digestion with the enzymes Escherichia coli 3-methyladenine-DNA glycosylase II, formamidopyrimidine-DNA glycosylase, Escherichia coli endonuclease III, or phage T4 UV endonuclease V did not increase the level of DNA breaks in NNKOAc-treated DNA. Detection of DNA damage using LMPCR was possible only when POB-DNA was 5'-phosphorylated prior to the LMPCR procedure. NNKOAc generated damage at all four bases with the decreasing order guanine>adenine>cytosine>thymine. In contrast to NNKOAc damage distribution patterns, those induced by N-nitroso(acetoxymethyl)methylamine, a methylating NNK analog, induced damage principally at G positions detectable by enzymatic means that did not require phosphorylation. Analysis of damage distribution patterns, reveals a high frequency of damage in the p53 gene in codons 241 and 245 and a lower frequency of damage in codon 248. We analyzed the 3' termini of the NNKOAc induced single-strand breaks using a (32)P-post-labeling assay or a nucleotide exchange reaction at the 3'-termini catalyzed by T4 DNA polymerase combined with endonuclease IV treatment. Both methods indicate that the 3' termini of the single-strand breaks are not hydroxyl groups and are blocked by an unknown chemical structure that is not recognized by endonuclease IV. These data are consistent with POB-phosphotriester hydrolysis leading to strand breaks in DNA. The POB-damage could be mutagenic because NNKOAc produces single-strand breaks with the products being a 5'-hydroxyl group and a 3'-blocking group and strand breaks. These results represent the first step in determining if NNK pyridyloxobutylates DNA with sequence specificity similar to those observed with other model compounds.
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PMID:Characterization and mapping of DNA damage induced by reactive metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at nucleotide resolution in human genomic DNA. 1167 38

Both the mutational status and the specific mutation of TP53 (p53) have been shown to impact both tumor prognosis and response to therapies. Molecular profiling of solid tumors is confounded by infiltrating wild-type cells, since normal DNA can interfere with detection of mutant sequences. Our objective was to identify TP53 mutations in 138 stage I-IV colorectal adenocarcinomas and liver metastases without first enriching for tumor cells by microdissection. To achieve this, we developed a harmonized protocol involving multiplex polymerase chain reaction/ligase detection reaction (PCR/LDR) with Universal DNA microarray analysis and endonuclease V/ligase mutation scanning. Sequences were verified using dideoxy sequencing. The harmonized protocol detected all 66 mutations. Dideoxy sequencing detected 41 out of 66 mutations (62%) using automated reading, and 59 out of 66 mutations (89%) with manual reading. Data analysis comparing colon cancer entries in the TP53 database (http://p53.curie.fr) with the results reported in this study showed that distribution of mutations and the mutational events were comparable.
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PMID:Harmonized microarray/mutation scanning analysis of TP53 mutations in undissected colorectal tumors. 1522 90

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