Strand selections resulting from the combined template-independent DNA synthesis and clamp activities of HIV-1 reverse transcriptase

Oz-Gleenberg, Iris; Hizi, Amnon

doi:10.1016/j.bbrc.2011.04.063

Cited by 8 publications

(12 citation statements)

References 15 publications

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“…All assays used in the present study and previously [7,8] measure the combined two‐step clamp/DNA polymerase activity, where the initial clamp is followed by DNA synthesis. We have used similar DNA polymerase activities for all tested RTs (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…Thus, the variations in the apparent combined clamp/polymerase activities of the tested RTs result solely from differences in clamp activity per se , as the only rate‐limiting step in the RT‐driven reaction. In the experiments described, we have used the clamp‐dependent DNA synthesis assay, as described previously in detail [7,8].…”

Section: Resultsmentioning

confidence: 99%

“…Thus, this activity potentially affects the efficiency of reverse transcription, allowing the RT‐associated DNA synthesis to bridge over nicks in the copied DNA or RNA templates while synthesizing DNA. The combined clamp activity and the capacity of some RTs to perform template‐independent blunt‐end DNA synthesis also support strand switches during DNA synthesis onto compatible acceptor strands [7,8].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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We have recently shown that reverse transcriptases (RTs) perform template switches when there is a very short (two-nucleotide) complementarity between the 3¢ ends of the primer (donor) strand and the DNA or RNA template acceptor strands ) Nucleic Acids Res 39, 1042-1053. These dinucleotide pairs are stabilized by RTs that are capable of 'clamping' together the otherwise unstable duplexes. This RT-driven stabilization of the micro-homology sequence promotes efficient DNA synthesis. In the present study, we have examined several factors associated with the sequence and structure of the DNA substrate that are critical for the clamp activity of RTs from human immunodeficiency virus type 1 (HIV-1), murine leukemia virus (MLV), bovine immunodeficiency virus (BIV) and the long terminal repeat retrotransposon Tf1. The parameters studied were the minimal complementarity length between the primer and functional template termini that sustains stable clamps, the effects of gaps between the two template strands on the clamp activity of the tested RTs, the effects of template end phosphorylations on the RT-associated clamp activities, and clamp activity with a long 'hairpin' double-stranded primer comprising both the primer and the complementary non-functional template strands. The results show that the substrate conditions for clamp activity of HIV-1 and MLV RTs are more stringent, while Tf1 and BIV RTs show clamp activity under less rigorous substrate conditions. These differences shed light on the dissimilarities in catalytic activities of RTs, and suggest that clamp activity may be a potential new target for anti-retroviral drugs.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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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.…”

mentioning

confidence: 99%

“…Therefore, when combined with their clamp activity, RTs can promote STs of the nascent DNA strands onto compatible acceptor strands. The combination between these RT functions depends on a variety of factors; namely, the extent of NTA activity, the stability of the terminal duplexes formed, the level of the RT's clamp activity, the bias of the dNTPs present, and the availability of proper acceptor strands (8).…”

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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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Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase

Pimentel

Upton

Collins

2022

Journal of Biological Chemistry

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Strand selections resulting from the combined template-independent DNA synthesis and clamp activities of HIV-1 reverse transcriptase

Cited by 8 publications

References 15 publications

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

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

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

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase

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