Determinants of adenine-mutagenesis in diversity-generating retroelements
| Autor: | Partho Ghosh, Andres Reyna, Sumit Handa, Timothy Wiryaman |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Purine
Models Molecular DNA Complementary Retroelements Guanine AcademicSubjects/SCI00010 Bordetella Protein Conformation Glycine Biology Arginine Catalysis chemistry.chemical_compound Models Complementary Information and Computing Sciences Complementary DNA Genetics Bacteriophages Computer Simulation chemistry.chemical_classification Base Sequence Cell-Free System Nucleic Acid Enzymes Adenine Mutagenesis Molecular RNA High-Throughput Nucleotide Sequencing Promoter RNA-Directed DNA Polymerase DNA Biological Sciences Reverse transcriptase Recombinant Proteins Amino acid chemistry Biochemistry Environmental Sciences Developmental Biology |
| Zdroj: | Nucleic Acids Research Nucleic acids research, vol 49, iss 2 |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | Diversity-generating retroelements (DGRs) vary protein sequences to the greatest extent known in the natural world. These elements are encoded by constituents of the human microbiome and the microbial ‘dark matter’. Variation occurs through adenine-mutagenesis, in which genetic information in RNA is reverse transcribed faithfully to cDNA for all template bases but adenine. We investigated the determinants of adenine-mutagenesis in the prototypical Bordetella bacteriophage DGR through an in vitro system composed of the reverse transcriptase bRT, Avd protein, and a specific RNA. We found that the catalytic efficiency for correct incorporation during reverse transcription by the bRT-Avd complex was strikingly low for all template bases, with the lowest occurring for adenine. Misincorporation across a template adenine was only somewhat lower in efficiency than correct incorporation. We found that the C6, but not the N1 or C2, purine substituent was a key determinant of adenine-mutagenesis. bRT-Avd was insensitive to the C6 amine of adenine but recognized the C6 carbonyl of guanine. We also identified two bRT amino acids predicted to nonspecifically contact incoming dNTPs, R74 and I181, as promoters of adenine-mutagenesis. Our results suggest that the overall low catalytic efficiency of bRT-Avd is intimately tied to its ability to carry out adenine-mutagenesis. |
| Databáze: | OpenAIRE |
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