Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The alpha-anomer of deoxyadenosine (alpha-dA) is a major adenine lesion produced by hydroxyl radicals in DNA. To assess its biochemical effects on DNA replication, alpha-dA was site-specifically incorporated into oligodeoxyribonucleotide templates using phosphoramidite chemistry. alpha-dA in the template constituted a transient block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (polI), but translesional synthesis occurred after prolonged incubation. Primer extension assays and Maxam-Gilbert sequencing of newly synthesized products revealed that alpha-dA directed not only incorporation of the correct nucleotide, dTMP, opposite the lesion but also misincorporation of dAMP and dCMP. dGMP was barely incorporated under these conditions. The order of the incorporation frequency at the alpha-dA site was affected by the nearest neighbor base pair 3' to the lesion. T7 and Taq DNA polymerases, as well as RAV-2 reverse transcriptase, showed a selectivity similar to that of PolI with respect to the nucleotide incorporation opposite alpha-dA, suggesting that the discrimination of nucleotides associated with alpha-dA is independent of the origin of DNA polymerases and is an intrinsic feature of the lesion. The mutational spectrum predicted for alpha-dA (i.e., A-->G transitions and A-->T transversions) is significantly different from those reported for other hydroxyl radical induced DNA lesions such as abasic sites or 7,8-dihydro-8-oxoguanine, both primarily directing misincorporation of A. Possible biological consequences and the mechanism of dNTP discrimination associated with alpha-dA are discussed.
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PMID:Replication of DNA templates containing the alpha-anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals. 800 79

The characterization of target nucleotides involved in the binding to DNA of 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000), a very potent genotoxic nitrofuran derivative, was investigated. Since R7000 undergoes metabolic activation prior to interacting with DNA, plasmids containing AT-rich and GC-rich sequences were devised and treated by R7000 in bacterial cells presenting nitroreductase activity. The nucleotide modifications to these homogeneous fragments that resulted from R7000 treatment were analyzed using the 'postlabeling' method. A preferential binding to the GC segment was demonstrated. Using a modification of the Maxam-Gilbert sequencing technique, it was demonstrated that activated R7000 creates alkali-labile phosphodiester bonds at the positions of guanines. In addition, the analysis of DNA replication-blocking properties of R7000 lesions was performed using avian myeloblastosis virus (AMV) reverse transcriptase as DNA polymerase. The termination of DNA replication occurred preferentially at the sites of guanine residues in the template strand, indicating that one nucleotide was inserted opposite a lesion. All these results indicate that guanine residues are the preferential sites of formation of R7000-DNA adducts.
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PMID:Determination of target nucleotides involved in 7-methoxy-2-nitro-naphtho[2,1-b]furan (R7000)-DNA adduct formation. 846 84


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