The Processivity of Telomerase: Insights from Kinetic Simulations and Analyses.

Clive R Bagshaw,Jendrik Hentschel,Michael D Stone

doi:10.3390/molecules26247532

Clive R Bagshaw, Jendrik Hentschel + Show 1 more

Open Access

https://doi.org/10.3390/molecules26247532

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Abstract

Telomerases are moderately processive reverse transcriptases that use an integral RNA template to extend the 3′ end of linear chromosomes. Processivity values, defined as the probability of extension rather than dissociation, range from about 0.7 to 0.99 at each step. Consequently, an average of tens to hundreds of nucleotides are incorporated before the single-stranded sDNA product dissociates. The RNA template includes a six nucleotide repeat, which must be reset in the active site via a series of translocation steps. Nucleotide addition associated with a translocation event shows a lower processivity (repeat addition processivity, RAP) than that at other positions (nucleotide addition processivity, NAP), giving rise to a characteristic strong band every 6th position when the product DNA is analyzed by gel electrophoresis. Here, we simulate basic reaction mechanisms and analyze the product concentrations using several standard procedures to show how the latter can give rise to systematic errors in the processivity estimate. Complete kinetic analysis of the time course of DNA product concentrations following a chase with excess unlabeled DNA primer (i.e., a pulse-chase experiment) provides the most rigorous approach. This analysis reveals that the higher product concentrations associated with RAP arise from a stalling of nucleotide incorporation reaction during translocation rather than an increased rate constant for the dissociation of DNA from the telomerase.

Highlights

Nucleic acid polymerases catalyze the elongation of a DNA or RNA strand, (N)n by the addition of one nucleotide at a time, derived from a nucleoside triphosphate substrate, according to Scheme 1: Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Scheme 1
After an appropriate incubation time, the reaction is stopped by the addition of EDTA and SDS, phenol-chloroform extracted and the DNA precipitated with ethanol, prior to separation by gel electrophoresis [6,7,12]
The product concentration in terms of DNA molecules needs to be adjusted according to the number of labeled nucleotides in the extended sequence

Summary

Introduction

Nucleic acid polymerases catalyze the elongation of a DNA or RNA strand, (N)n by the addition of one nucleotide at a time, derived from a nucleoside triphosphate substrate, according to Scheme 1: Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Scheme 1. The extension of a nucleic acid chain by nucleotide addition. The correct nucleotide for incorporation is selected according to the sequence of a complementary template strand. After the addition of a nucleotide, the newly formed duplex product moves out of the active site to make way for free base in the template sequence. The duplex usually remains attached to the polymerase during this translocation. Most polymerases are highly processive and many hundreds or thousands of bases may be incorporated before the product nucleic acid dissociates from the polymerase

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