Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity

Zúñiga, Roberto Ariel Abeldaño; Sengupta, Sonali; Snyder, Christine; León, Óscar; Roth, Monica J.

doi:10.1093/protein/gzh071

Cited by 6 publications

(3 citation statements)

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“…This is in marked contrast to the RNase H of RTs. A protein derived from MuLV RT containing only the RNase H domain retains activity but loses its specificity (29), while in HIV-1 RT the RNase H domain relies on other portions of the protein for activity (30,31). …”

Section: Discussionmentioning

confidence: 99%

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Gaidamakov

Gorshkova²,

Schuck³

et al. 2005

Nucleic Acids Research

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Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.

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Section: Discussionmentioning

confidence: 99%

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Gaidamakov

Gorshkova²,

Schuck³

et al. 2005

Nucleic Acids Research

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“…Taken together, these results indicate that the essential catalytic properties of the RNase H domain were maintained in p51-G-TCR. (47). The p51-G-TCR Mg 2ϩ -dependent RNase H construct differs from the prior TCCR construct by incorporating the p66 thumb and MGBT as well as the p51 finger-palm domain.…”

Section: Resultsmentioning

confidence: 99%

“…Previous attempts to express the HIV-1 RNase H as a catalytically active domain have been possible only in the presence of Mn 2ϩ , and many of these attempts required an alternative substrate binding domain (16,21,39,40,42,47). The role of metal binding in the RNase H domain of HIV has been a controversial area.…”

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confidence: 99%

Expression of an Mg ²⁺ -Dependent HIV-1 RNase H Construct for Drug Screening

Farias

Vargas

Castillo

et al. 2011

Antimicrob Agents Chemother

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A single polypeptide of the HIV-1 reverse transcriptase that reconstituted Mg 2؉ -dependent RNase H activity has been made. Using molecular modeling, the construct was designed to encode the p51 subunit joined by a linker to the thumb (T), connection (C), and RNase H (R) domains of p66. This p51-G-TCR construct was purified from the soluble fraction of an Escherichia coli strain, MIC2067(DE3), lacking endogenous RNase HI and HII. The p51-G-TCR RNase H construct displayed Mg 2؉ -dependent activity using a fluorescent nonspecific assay and showed the same cleavage pattern as HIV-1 reverse transcriptase (RT) on substrates that mimic the tRNA removal required for second-strand transfer reactions. The mutant E706Q (E478Q in RT) was purified under similar conditions and was not active. The RNase H of the p51-G-TCR RNase H construct and wild type HIV-1 RT had similar K m s for an RNA-DNA hybrid substrate and showed similar inhibition kinetics to two known inhibitors of the HIV-1 RT RNase H.

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Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2

2015

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Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity

Cited by 6 publications

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Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Expression of an Mg ²⁺ -Dependent HIV-1 RNase H Construct for Drug Screening

Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2

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Expression of the C-terminus of HIV-1 reverse transcriptase p66 and p51 subunits as a single polypeptide with RNase H activity

Cited by 6 publications

References 0 publications

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Expression of an Mg 2+ -Dependent HIV-1 RNase H Construct for Drug Screening

Enzymatic Activities of RNase H Domains of HIV-1 Reverse Transcriptase with Substrate Binding Domains of Bacterial RNases H1 and H2

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Expression of an Mg ²⁺ -Dependent HIV-1 RNase H Construct for Drug Screening