Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase

Abstract

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Substrate Specificity and Kinetics of RNA Hydrolysis by SARS-CoV-2 NSP10/14 Exonuclease

“…We began by choosing three RNA substrates on which to measure excision. Most proofreading exonucleases function on at least partially single-stranded nucleic acids arising from the melting of duplex DNA or RNA at the polymerase active site to transfer the 3′-end of the primer into the exonuclease active site. ,,− Our data show that, unlike other proofreading exonucleases, neither single-stranded RNA nor double-stranded RNA containing a mismatch is a preferred substrate, exhibiting rates of hydrolysis 45- to 125-fold slower than rates of excision on correctly base-paired RNA. This observation may imply that the RNA duplex must dissociate completely from the polymerase active site and both strands of the RNA must rebind at the exonuclease active site without tethering the template strand in the polymerase active site, as previously suggested .…”

“…Double-stranded RNA containing either correctly base-paired or mispaired 3′-terminal primer strands represents RNA replication intermediates that would be continuously formed by the RdRp during genome replication and possibly excised by the exonuclease. For well-characterized proofreading exonucleases, single-stranded nucleic acids are the preferred substrates. , When primer extension at the polymerase active site stalls, the primer strand of duplex DNA can melt away from the complementary template strand and enter the exonuclease active site where the 3′-terminal base is rapidly excised …”

“…High-quality single turnover kinetic analysis of the exonuclease complex in conjunction with the RdRp and other viral nonstructural proteins are required to address these questions about whether the substrate specificity of the NSP10/14 complex changes in conjunction with other binding partners. Moreover, the critical role of the proofreading exonuclease will rely on the relative rates of primer extension versus excision from a single multienzyme complex containing both activities, as shown for DNA polymerases. , …”

Chimeric DNA byproducts in strand displacement amplification using the T7 replisome

“…In general, linear amplification methods, in which the product DNA cannot act as a substrate, have slower kinetics and limited sensitivity compared to exponential methods like PCR or MDA [ 7 ]. Linear amplification has been using in a single-cell sequencing technique to avoid bias introduced by PCR and increase sequencing accuracy [ 47 ]; however, Δ28 gp5 has a reduced fidelity relative to WT gp5 owing to the mutations in the proof-reading exonuclease domain [ 48 ]. A possibility is that the WT T7 replisome could be used with a non-specific nicking endonuclease, e.g.…”

Kinetics of DNA strand transfer between polymerase and proofreading exonuclease active sites regulates error correction during high-fidelity replication

“…We then used this variant to characterize the conformational dynamics that govern correct nucleotide incorporation ( 9 ) and compared that with mismatched nucleotide incorporation ( 10 ) to define the mechanistic basis for the extraordinary fidelity of this enzyme. We also previously characterized the DNA substrate specificity for the proofreading exo of T7 DNA pol using rapid-quench methods and showed that the rate of mismatch excision increased with the single-stranded nature of the DNA substrate; that is, ssDNA was hydrolyzed the fastest, whereas the 3′-terminal base in duplex DNA was removed at a rate that increased with the number of mismatched base pairs ( 11 ). Surprisingly, we found that a mismatch buried by a single correct base pair was removed much more efficiently than a single terminal mismatch, demonstrating that the enzyme affords multiple opportunities to remove a mismatch.…”