Pyrophosphate-Mediated Repair of Damaged and Mismatched RNA by a Polymerase Ribozyme

Abstract

AbstractPrior to the emergence of the contemporary biosphere, the early Earth is likely to have progressed through an RNA-based world. In such an evolving world, it would have been critical to have a system of genome replication that allowed heritable information to be transferred with an early catalyst, such as an RNA polymerase ribozyme. Though substantial effort has gone into evolving such a polymerase, many variants suffer from premature termination and low fidelity, resulting in low yields of full-length sequences and small quantities of active sequences, respectively. The ability to replicate and maintain a genome at a higher fidelity would lend an evolutionary advantage to an early evolvable system. Cells today achieve this through a variety of means, including complex damage repair mechanisms and proofreading pathways, but such dedicated machinery would likely not exist in an early evolving world. However, if an early polymerase possessed repair activity under specific conditions, replication would have been improved. Here we demonstrate the first example of ribozyme-mediated repair of damaged and mismatched RNA sequences. Under conditions of high pyrophosphate concentrations, we show that a polymerase ribozyme can repair RNA sequences terminated in a 2′ −3′ cyclic phosphate, a mismatch, or both, to allow further polymerization along a template. In many cases, the fidelity of the repaired RNA extension is significantly higher than priming from a mismatched base-pair; this observation suggests that mutations may have an allosteric effect on the fidelity of downstream replication. Finally, we show that the pyrophosphorolysis reaction directly yields a nucleoside triphosphate, providing the first evidence for ribozyme-catalyzed production of ATP. Increased fidelity and processivity of polymerization advances the longstanding goal of developing a self-replicating polymerase ribozyme.