Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 294, Issue 51, Pages 19764-19784Publisher
ELSEVIER
DOI: 10.1074/jbc.RA119.011337
Keywords
chemical biology; DNA sequencing; enzyme kinetics; retrovirus; reverse transcription; RNA; RNA virus; structure?function; transposable element (TE); viral polymerase; group II intron reverse transcriptase; non-templated nucleotide addition; RNA sequencing; RNA-dependent RNA polymerase; thermostable group II intron reverse transcriptase
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Funding
- National Institutes of Health [R01 GM37949, R35 GM131777]
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The reverse transcriptases (RTs) encoded by mobile group II introns and other non-LTR retroelements differ from retroviral RTs in being able to template-switch efficiently from the 5? end of one template to the 3? end of another with little or no complementarity between the donor and acceptor templates. Here, to establish a complete kinetic framework for the reaction and to identify conditions that more efficiently capture acceptor RNAs or DNAs, we used a thermostable group II intron RT (TGIRT; GsI?IIC RT) that can template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3?-DNA overhang end. We found that the rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3?-DNA overhang complementary to the 3? nucleotide of the acceptor RNA, suggesting a role for nontemplated nucleotide addition of a complementary nucleotide to the 3? end of cDNAs synthesized from natural templates. Longer 3?-DNA overhangs progressively decreased the template-switching rate, even when complementary to the 3? end of the acceptor template. The reliance on only a single bp with the 3? nucleotide of the acceptor together with discrimination against mismatches and the high processivity of group II intron RTs enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3? end. We discuss the possible biological functions of the template-switching activity of group II intron- and other non-LTR retroelement?encoded RTs, as well as the optimization of this activity for adapter addition in RNA- and DNA-Seq protocols.
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