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

5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C-->T transitions, resulting from spontaneous deamination of mC to T. The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesis via G-->A transitions. Stimulation of the G-->A mutational pathway could involve preferential misincorporation of dAMP opposite template mC compared to C. To investigate this possibility, we synthesized a sequence containing mC at a defined template location. We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity. Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase. AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite mC than C. Favored extension of a 5-MeC.A over C.A mispair was also observed with a difference of about 3-fold. In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either mC or C sites or when extending mispairs involving mC and C. Incorporation of dAMP opposite either C or mC was barely detectable using pol alpha, although pol alpha has been observed to form A.C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-->A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.
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PMID:A comparison of the fidelity of copying 5-methylcytosine and cytosine at a defined DNA template site. 138 39

5-Methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), two of the best-studied DNA modifications, play crucial roles in normal development and disease in mammals. Although 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) have also been identified in RNA, their distribution and biological function in RNA remain largely unexplored, due to the lack of suitable sequencing methods. Here, we report a base-resolution sequencing method for hm5C in RNA. We applied the selective oxidation of hm5C to trihydroxylated-thymine (thT) mediated by peroxotungstate. thT was subsequently converted to T during cDNA synthesis using a thermostable group II intron reverse transcriptase (TGIRT). Base-resolution analysis of the hm5C sites in RNA was performed using Sanger sequencing. Furthermore, in combination with the TET enzyme oxidation of m5C to hm5C in RNA, we expand the use of peroxotungstate oxidation to detect m5C in RNA at base-resolution. By using this method, we confirmed three known m5C sites in human tRNA, demonstrating the applicability of our method in analyzing real RNA samples.
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PMID:Bisulfite-free and base-resolution analysis of 5-methylcytidine and 5-hydroxymethylcytidine in RNA with peroxotungstate. 3072 49