Mirror-Image Thymidine Discriminates against Incorporation of Deoxyribonucleotide Triphosphate into DNA and Repairs Itself by DNA Polymerases

Abstract

DNA polymerases are known to recognize preferably d-nucleotides over l-nucleotides during DNA synthesis. Here, we report that several general DNA polymerases catalyze polymerization reactions of nucleotides directed by the DNA template containing an l-thymidine (l-T). The results display that the 5'-3' primer extension of natural nucleotides get to the end at chiral modification site with Taq and Phanta Max DNA polymerases, but the primer extension proceeds to the end of the template catalyzed by Deep Vent (exo), Vent (exo), and Therminator DNA polymerases. Furthermore, templating l-nucleoside displays a lag in the deoxyribonucleotide triphosphate (dNTP) incorporation rates relative to natural template by kinetics analysis, and polymerase chain reactions were inhibited with the DNA template containing two or three consecutive l-Ts. Most interestingly, no single base mutation or mismatch mixture corresponding to the location of l-T in the template was found, which is physiologically significant because they provide a theoretical basis on the involvement of DNA polymerase in the effective repair of l-T that may lead to cytotoxicity.

Therminator DNA Polymerase: Modified Nucleotides and Unnatural Substrates

“…In addition to incorporating modified nucleotides on a natural DNA template, Therminator DNA Polymerase accommodates modifications in the template strand. For example, Xiao et al demonstrate high fidelity synthesis of dATP across from mirror image L-thymidine in the template strand using Therminator DNA Polymerase (Xiao et al, 2017). Therefore, the ability of Therminator DNA Polymerase to synthesize modifications on both the primer and template strands will increase the diversity of modifications that can be introduced into substrates.…”

Compatibility and Fidelity of Mirror-Image Thymidine in Transcription Events by T7 RNA Polymerase

“… 35 Previous studies have shown that l -nucleotide residue in heterochiral duplexes retains its selectivity toward the complementary d -nucleotide residue. 17 , 36 Considering that T7 RNAP can incorporate NTPs opposite l -T in the templates into transcripts with different efficiency, we therefore checked the fidelity of the T7 RNAP when it encountered l -T-containing templates.…”

“…Recently, we reported that natural nucleotides could be incorporated into the growing chain opposite of the l -thymidine ( l -T) unit in the DNA template by B family DNA polymerases (Therminator, Vent [exo − ], and Deep Vent [exo − ] DNA polymerase) and elongate the primer with self-repair. 17 Based on the results of primer extension, further investigation on the role of mirror-image thymidine in bypassing these lesions in RNA transcription reactions was necessary.…”

“…Although DNA replication studies with A and B family DNA polymerases have been carried out for l -T insertion, 17 our transcriptional systems were used to estimate the degrees to which the lesion impedes RNA transcription and whether it induces mutations. In this study, we have introduced l -T into different sites of template strands and non-template strands, and then examined the processivity of transcription with the modified templates catalyzed by T7 RNAP.…”

Mutation Analysis of L‐Thymidine‐Induced Replication Products Using a Restriction Enzyme–Mediated Assay

“…In the steps below, L-T is introduced into nucleic acids according to a published method (Xiao et al, 2017). The antiviral drug Telbivudine corresponds to 2 -deoxy-L-thymidine (Yan, Qiao, Zhang, Wang, & Gou, 2018).…”