Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases

Oz-Gleenberg, Iris; Herzig, Eytan; Voronin, Nickolay; Hizi, Amnon

doi:10.1111/j.1742-4658.2012.08570.x

Cited by 13 publications

(19 citation statements)

References 16 publications

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“…Biochemical studies showed that a related activity of HIV-1 RT (referred to as "clamping") requires at least 2 bps between the 3Ј end of the cDNA and the acceptor template, one of which must be a GC or CG bp (17). Other retroviral and LTR retroelement RTs also required 2 bps for maximal clamping activity, but in some cases they showed weak activity (24 -36%) with a single bp (18).…”

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confidence: 99%

Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

Lentzsch

Yao

Russell

et al. 2019

Journal of Biological Chemistry

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Edited by Karin Musier-Forsyth This work was supported by National Institutes of Health Grants R01 GM37949 (to A. M. Lambowitz) and R35 GM131777 (to R. R.). Thermostable group II intron reverse transcriptase enzymes and methods for their use are the subject of patents and patent applications that have been licensed by the University of Texas and East Tennessee State University to InGex, LLC. A. M. Lambowitz, some former and present members of the Lambowitz laboratory, and the University of Texas are minority equity holders in InGex, LLC, and receive royalty payments from the sale of TGIRT enzymes and kits employing TGIRT template-switching activity for RNA-seq adapter addition and from the sublicensing of intellectual property to other companies. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S10 and Tables S1-S3. RNA-Seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE138200.

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confidence: 99%

Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

Lentzsch

Yao

Russell

et al. 2019

Journal of Biological Chemistry

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“…5), increasing the time window for sampling potential acceptor templates for complementarity. Longer base-pairing interactions assist template switching by retroviral and LTR-retrotransposon RTs (18), but the opposite is true for GsI-IIC RT, with overhang lengths beyond 1-nt decreasing the efficiency of template switching ( Fig. 6 and Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Template switching by a retroviral RT is also employed for RNA-seq adapter addition in a method called SMART-Seq (Switching Mechanism At the 5' end of the RNA Transcript sequencing), which utilizes the ability of Moloney murine leukemia virus RT to add nontemplated C residues to the 3' end of a completed cDNA, which can then base pair and enable template switching to the 3' end of an adapter DNA oligonucleotide ending with G residues (4,7,16). Biochemical studies showed that efficient template switching by HIV-1 RT requires at least two base pairs between the 3' end of the cDNA and the acceptor template, one of which must be a GC or CG base pair (17,18).…”

Section: Introductionmentioning

confidence: 99%

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

Yao

Russell

et al. 2019

Preprint

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Group II intron reverse transcriptase template switching 2 ABSTRACTThe reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retroelements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT; GsI-IIC RT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. 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 non-templated nucleotide addition of a complementary nucleotide to the 3' end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. The reliance on a single base pair with the 3' nucleotide of the acceptor together with discrimination against mismatches and the high processivity of the enzyme enable synthesis of full-length DNA copies of nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTRretroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.

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“…New in vitro evidence, presented recently by us, show that RTs can perform also template switches with even a very short (1 to 2 nt) complementarity between the 3= ends of the primer donor strand and the DNA or RNA template acceptor strands (6)(7)(8). These tiny duplexes are markedly stabilized thermodynamically by the RT that "clamps" together the duplex structures that are otherwise very unstable.…”

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confidence: 99%

“…This new clamp function is performed, though to variable extents, by all RTs tested but not by cellular DNA polymerases, suggesting that it is potentially essential to retroviral RTN (6). Interestingly, RTs, other than those of HIV and MLV, can also form a stable clamp with even a single nucleotide complementarity between the donor and acceptor strands, and these RTs can tolerate even short 1-to 2-nt gaps between the functional template and the adjacent strand (7).…”

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confidence: 99%

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

Herzig

Voronin

Kucherenko

et al. 2015

J Virol

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The process of reverse transcription (RTN) in retroviruses is essential to the viral life cycle. This key process is catalyzed exclusively by the viral reverse transcriptase (RT) that copies the viral RNA into DNA by its DNA polymerase activity, while concomitantly removing the original RNA template by its RNase H activity. During RTN, the combination between DNA synthesis and RNA hydrolysis leads to strand transfers (or template switches) that are critical for the completion of RTN. The balance between these RT-driven activities was considered to be the sole reason for strand transfers. Nevertheless, we show here that a specific mutation in HIV-1 RT (L92P) that does not affect the DNA polymerase and RNase H activities abolishes strand transfer. There is also a good correlation between this complete loss of the RT's strand transfer to the loss of the DNA clamp activity of the RT, discovered recently by us. This finding indicates a mechanistic linkage between these two functions and that they are both direct and unique functions of the RT (apart from DNA synthesis and RNA degradation). Furthermore, when the RT's L92P mutant was introduced into an infectious HIV-1 clone, it lost viral replication, due to inefficient intracellular strand transfers during RTN, thus supporting the in vitro data. As far as we know, this is the first report on RT mutants that specifically and directly impair RT-associated strand transfers. Therefore, targeting residue Leu92 may be helpful in selectively blocking this RT activity and consequently HIV-1 infectivity and pathogenesis. IMPORTANCEReverse transcription in retroviruses is essential for the viral life cycle. This multistep process is catalyzed by viral reverse transcriptase, which copies the viral RNA into DNA by its DNA polymerase activity (while concomitantly removing the RNA template by its RNase H activity). The combination and balance between synthesis and hydrolysis lead to strand transfers that are critical for reverse transcription completion. We show here for the first time that a single mutation in HIV-1 reverse transcriptase (L92P) selectively abolishes strand transfers without affecting the enzyme's DNA polymerase and RNase H functions. When this mutation was introduced into an infectious HIV-1 clone, viral replication was lost due to an impaired intracellular strand transfer, thus supporting the in vitro data. Therefore, finding novel drugs that target HIV-1 reverse transcriptase Leu92 may be beneficial for developing new potent and selective inhibitors of retroviral reverse transcription that will obstruct HIV-1 infectivity. R everse transcription (RTN) is a critical step in the life cycle of all retroviruses and the related long terminal repeat (LTR) retrotransposons. This complex multistep process is performed by a single enzyme, the retroviral reverse transcriptase (RT) that copies the viral plus strand RNA into integration competent double-stranded viral DNA (1-3). To perform RTN, RTs have two activities. These are the DNA polymerase activity, which co...

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Substrate variations that affect the nucleic acid clamp activity of reverse transcriptases

Cited by 13 publications

References 16 publications

Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

Template-switching mechanism of a group II intron-encoded reverse transcriptase and its implications for biological function and RNA-Seq

Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

A Novel Leu92 Mutant of HIV-1 Reverse Transcriptase with a Selective Deficiency in Strand Transfer Causes a Loss of Viral Replication

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