دورية أكاديمية
Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.
| العنوان: | Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning. |
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| المؤلفون: | Potapov V; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA., Krudup S; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA.; École Supérieure de Biotechnologie de Strasbourg, 67400 Strasbourg, France., Maguire S; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA., Unlu I; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA., Guan S; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA., Buss JA; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA., Smail BA; Department of Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA., van Eeuwen T; Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, New York 10065, USA., Taylor MS; Department of Pathology, Mass General Brigham and Harvard Medical School, Boston, Massachusetts 02114, USA., Burns KH; Department of Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA.; Department of Pathology, Mass General Brigham and Harvard Medical School, Boston, Massachusetts 02114, USA., Ong JL; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA ong@neb.com rtrachman@neb.com., Trachman RJ 3rd; New England Biolabs, Inc., Ipswich, Massachusetts 01938, USA ong@neb.com rtrachman@neb.com. |
| المصدر: | RNA (New York, N.Y.) [RNA] 2024 Aug 16; Vol. 30 (9), pp. 1246-1258. Date of Electronic Publication: 2024 Aug 16. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Cold Spring Harbor Laboratory Press Country of Publication: United States NLM ID: 9509184 Publication Model: Electronic Cited Medium: Internet ISSN: 1469-9001 (Electronic) Linking ISSN: 13558382 NLM ISO Abbreviation: RNA Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2003->: Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press Original Publication: New York, NY : Cambridge University Press, c1995- |
| مواضيع طبية MeSH: | RNA-Directed DNA Polymerase*/metabolism , RNA-Directed DNA Polymerase*/genetics , DNA-Directed RNA Polymerases*/metabolism , DNA-Directed RNA Polymerases*/genetics, Humans ; Viral Proteins/genetics ; Viral Proteins/metabolism ; Evolution, Molecular ; Mutation ; DNA, Complementary/genetics |
| مستخلص: | Direct methods for determining the fidelity of DNA polymerases are robust, with relatively little sample manipulation before sequencing. In contrast, methods for measuring RNA polymerase and reverse transcriptase fidelities are complicated by additional preparation steps that introduce ambiguity and error. Here, we describe a sequencing method, termed Roll-Seq, for simultaneously determining the individual fidelities of RNA polymerases and reverse transcriptases (RT) using Pacific Biosciences single molecule real-time sequencing. By using reverse transcriptases with high rolling-circle activity, Roll-Seq generates long concatemeric cDNA from a circular RNA template. To discern the origin of a mutation, errors are recorded and determined to occur within a single concatemer (reverse transcriptase error) or all concatemers (RNA polymerase error) over the cDNA strand. We used Roll-Seq to measure the fidelities of T7 RNA polymerases, a Group II intron-encoded RT (Induro), and two LINE RTs ( Fasciolopsis buski R2-RT and human LINE-1). Substitution rates for Induro and R2-RT are the same for cDNA and second-strand synthesis while LINE-1 has 2.5-fold lower fidelity when performing second-strand synthesis. Deletion and insertion rates increase for all RTs during second-strand synthesis. In addition, we find that a structured RNA template impacts fidelity for both RNA polymerase and RT. The accuracy and precision of Roll-Seq enable this method to be applied as a complementary analysis to structural and mechanistic characterization of RNA polymerases and reverse transcriptases or as a screening method for RNAP and RT fidelity. (© 2024 Potapov et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.) |
| فهرسة مساهمة: | Keywords: RNA polymerase; fidelity; reverse transcriptase; sequencing |
| المشرفين على المادة: | EC 2.7.7.49 (RNA-Directed DNA Polymerase) EC 2.7.7.6 (DNA-Directed RNA Polymerases) EC 2.7.7.- (bacteriophage T7 RNA polymerase) 0 (Viral Proteins) 0 (DNA, Complementary) |
| تواريخ الأحداث: | Date Created: 20240628 Date Completed: 20240816 Latest Revision: 20240821 |
| رمز التحديث: | 20240821 |
| مُعرف محوري في PubMed: | PMC11331410 |
| DOI: | 10.1261/rna.080002.124 |
| PMID: | 38942481 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 1469-9001 |
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| DOI: | 10.1261/rna.080002.124 |