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Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase

Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribut...

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Autores principales: Lentzsch, Alfred M., Stamos, Jennifer L., Yao, Jun, Russell, Rick, Lambowitz, Alan M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363836/
https://www.ncbi.nlm.nih.gov/pubmed/34280434
http://dx.doi.org/10.1016/j.jbc.2021.100971
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author Lentzsch, Alfred M.
Stamos, Jennifer L.
Yao, Jun
Russell, Rick
Lambowitz, Alan M.
author_facet Lentzsch, Alfred M.
Stamos, Jennifer L.
Yao, Jun
Russell, Rick
Lambowitz, Alan M.
author_sort Lentzsch, Alfred M.
collection PubMed
description Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template–DNA primer heteroduplex with a 1-nt 3′-DNA overhang. The structure showed that the 3′ end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non–long terminal repeat–retroelement RTs and the RT fingertips loop, with the 3′ nucleotide of the acceptor base paired to the 1-nt 3′-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non–long terminal repeat–retroelement RTs and viral RNA–dependent RNA polymerases.
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spelling pubmed-83638362021-08-23 Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase Lentzsch, Alfred M. Stamos, Jennifer L. Yao, Jun Russell, Rick Lambowitz, Alan M. J Biol Chem Research Article Reverse transcriptases (RTs) can switch template strands during complementary DNA synthesis, enabling them to join discontinuous nucleic acid sequences. Template switching (TS) plays crucial roles in retroviral replication and recombination, is used for adapter addition in RNA-Seq, and may contribute to retroelement fitness by increasing evolutionary diversity and enabling continuous complementary DNA synthesis on damaged templates. Here, we determined an X-ray crystal structure of a TS complex of a group II intron RT bound simultaneously to an acceptor RNA and donor RNA template–DNA primer heteroduplex with a 1-nt 3′-DNA overhang. The structure showed that the 3′ end of the acceptor RNA binds in a pocket formed by an N-terminal extension present in non–long terminal repeat–retroelement RTs and the RT fingertips loop, with the 3′ nucleotide of the acceptor base paired to the 1-nt 3′-DNA overhang and its penultimate nucleotide base paired to the incoming dNTP at the RT active site. Analysis of structure-guided mutations identified amino acids that contribute to acceptor RNA binding and a phenylalanine residue near the RT active site that mediates nontemplated nucleotide addition. Mutation of the latter residue decreased multiple sequential template switches in RNA-Seq. Our results provide new insights into the mechanisms of TS and nontemplated nucleotide addition by RTs, suggest how these reactions could be improved for RNA-Seq, and reveal common structural features for TS by non–long terminal repeat–retroelement RTs and viral RNA–dependent RNA polymerases. American Society for Biochemistry and Molecular Biology 2021-07-17 /pmc/articles/PMC8363836/ /pubmed/34280434 http://dx.doi.org/10.1016/j.jbc.2021.100971 Text en © 2021 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Article
Lentzsch, Alfred M.
Stamos, Jennifer L.
Yao, Jun
Russell, Rick
Lambowitz, Alan M.
Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title_full Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title_fullStr Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title_full_unstemmed Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title_short Structural basis for template switching by a group II intron–encoded non-LTR-retroelement reverse transcriptase
title_sort structural basis for template switching by a group ii intron–encoded non-ltr-retroelement reverse transcriptase
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363836/
https://www.ncbi.nlm.nih.gov/pubmed/34280434
http://dx.doi.org/10.1016/j.jbc.2021.100971
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