Discrete measurements of RNA polymerase and reverse transcriptase fidelity reveal evolutionary tuning.
RNA
; 30(9): 1246-1258, 2024 Aug 16.
Article
en En
| MEDLINE
| ID: mdl-38942481
ABSTRACT
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.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Polimerasas Dirigidas por ADN
/
ADN Polimerasa Dirigida por ARN
Límite:
Humans
Idioma:
En
Revista:
RNA
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos