Purification and characterization of the RNase H domain of HIV‐1 reverse transcriptase expressed in recombinant <i>Escherichia coli</i>

Becerra, S. Patricia; Clore, G. Marius; Gronenborn, Angela M.; Karlström, Anders R.; Stahl, Stephen J.; Wilson, Samuel H.; Wingfield, Paul T.

doi:10.1016/0014-5793(90)81238-j

Cited by 55 publications

(35 citation statements)

References 18 publications

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“…The p15 RNase H domain of HIV-1 exhibits structural similarity to Escherichia coli RNase H (36). But unlike the E. coli RNase H, which cleaves the RNA component of RNA/DNA duplexes randomly, the isolated p15 of RT has been shown to be devoid of RNase H function (36,37). In combination with the data available in the literature (18, 20, 21, 36 -42), our results strongly support the notion that in the absence of structural elements responsible for RT positioning on the polynucleotide substrate, the RNase H active site is rendered inactive.…”

Section: Discussionsupporting

confidence: 84%

Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

Palaniappan¹,

Wisniewski²,

Jacques

et al. 1997

Journal of Biological Chemistry

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Human immunodeficiency virus (HIV) DNA synthesis is accompanied by degradation of genomic RNA by the RNase H of reverse transcriptase (RT). Two different modes of RNase H activity appear necessary for complete RNA removal. In one, occurring during minus strand synthesis, positioning of the RNase H is determined by binding of the polymerase active site to the DNA 3-end. In the other, used for removal of remaining RNA fragments, positioning of RT for RNase H-directed cleavage is determined by the RNA 5-ends. We attempted to identify RT amino acids responsible for these modes of positioning. Twelve RT mutants, each with one alanine replacement in residues 224 to 235, known as the primer grip region, were examined for catalytic abilities. Six of the examined primer grip mutants, although distant from the RNase H active site were altered in their ability to cleave RNA. The mutants P226A, F227A, G231A, Y232A, E233A, and H235A failed to perform RNA 5-end-directed RNase H cleavage in heparin-challenged reactions. The last four mutants also lacked DNA synthesis and DNA 3-end-directed RNase H cleavage activities in challenged reactions. Since mutants P226A and F227A carried out these latter reactions normally, these two residues specifically influence 5-RNA-directed RNase H catalysis. Human immunodeficiency virus, type 1 (HIV-1)1 is the causative agent of AIDS. During replication, the virally encoded reverse transcriptase catalyzes the conversion of the singlestranded RNA genome to a double-stranded DNA genome. HIV-RT catalyzes RNA-directed DNA synthesis, DNA-directed DNA synthesis, RNase H, strand displacement, and strand transfer activities (1). The RNA-directed DNA polymerase activity is essential for the formation of minus strand DNA from plus strand genomic RNA. RNase H activity is required for the removal of the RNA portion of the RNA/DNA hybrid formed during minus strand DNA synthesis. Biochemical and structural measurements show that the DNA polymerase and RNase H active sites are separated by a distance of about 18 nt along the template (2-8). Estimates vary from 14 to 20 nt in biochemical studies depending on the sequence of the nucleic acid employed. When the polymerase active site was bound at the 3Ј-OH of a DNA primer on an RNA template, this positioning determined the first site of cleavage of the template at the distance separated by the active sites (2, 3). Hence, this was termed the polymerase-dependent mode of RNase H-directed cleavage. Cleavage at other positions was termed polymeraseindependent. This latter class includes secondary cleavages that occur near the polymerase-dependent cleavage site but closer to the DNA 3Ј-end. Following the initial cleavage, the RT displays a 3Ј 3 5Ј directional processive RNase H activity (9 -12).As suggested by our biochemical studies, cleavage of the plus strand RNA is not completed by the RT that synthesizes the minus DNA strand (13). Fragments of RNA 13-45 nt in length are left behind that stay annealed to the newly synthesized DNA. Two of these are the polypurine t...

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Section: Discussionsupporting

confidence: 84%

Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

Palaniappan¹,

Wisniewski²,

Jacques

et al. 1997

Journal of Biological Chemistry

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“…3A). RNH102 was inactive under these conditions, and as has been observed by others (10,(37)(38)(39), the wild-type HIV-1 domain was also inactive. Very different results were obtained when Mn2+ was substituted for Mg2+ as the divalent cation (Fig.…”

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confidence: 50%

Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.

Keck

Marqusee

1995

Proc. Natl. Acad. Sci. U.S.A.

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“…The importance of this region for substrate binding has been demonstrated in several studies. The RNase HI domain isolated from HIV-1 reverse transcriptase is enzymatically inactive (13,14), whereas that from murine leukemia virus reverse transcriptase, which has a part of this region, is active (15,16). Site-directed mutagenesis indicated that the positively charged residues in this region are important for substrate binding (17).…”

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confidence: 99%

Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

Haruki¹,

Noguchi²,

Kanaya³

et al. 1997

Journal of Biological Chemistry

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To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcore). The k on and k off values for the binding of the enzyme to the 36-bp substrate were 1. 5 (5) in the catalytic function was established by site-directed mutagenesis for the catalytic function of the enzyme. Two alternative mechanisms have been proposed: one is a two-metal ion mechanism (6) and the other is a carboxyl-hydroxyl relay mechanism (4, 7-9).X-ray crystallographic analyses of E. coli RNase HI (6, 10, 11) and the RNase H domain of HIV-1 1 reverse transcriptase (12) showed that these RNases H have a similar structural topology, with the exception of the presence of a handle (6) or basic protrusion region (11) in E. coli RNase HI. The importance of this region for substrate binding has been demonstrated in several studies. The RNase HI domain isolated from HIV-1 reverse transcriptase is enzymatically inactive (13,14), whereas that from murine leukemia virus reverse transcriptase, which has a part of this region, is active (15, 16). Site-directed mutagenesis indicated that the positively charged residues in this region are important for substrate binding (17). Incorporation of the basic protrusion of E. coli RNase HI at the equivalent position of the RNase H domain of HIV-1 reverse transcriptase resulted in the production of the active HIV-1 RNase H domain (18,19 However, it is not fully understood how RNase H binds to its substrate. A kinetic study using synthetic nucleosides with modifications of their 2Ј-hydroxyl groups revealed the importance of the 2Ј-hydroxyl group of the nucleoside on the 3Ј-side of the cleaved phosphodiester and that of the second nucleoside 5Ј to the cleaved phosphodiester for hydrogen bonding (20). Models for the binding of the enzyme to an RNA-DNA hybrid have been proposed based upon computer docking of the structure of E. coli RNase HI (free from its substrate) with an RNA-DNA hybrid whose structure was assumed to be an A form (6, 11), was found by NMR to be an A form (7), or that was neither A nor B (21). In these models, the RNA strand upstream of the cleavage site interacts with the enzyme. None of these models assumes that either the enzyme or the substrate alters its conformation upon binding.Recently, kinetic analyses using RNA-DNA hybrids, under conditions in which the hybrid was cleaved at a unique site (22), suggest that DNA residues complementary to the RNA residues located six or seven residues upstream of the cleavage site interact with the basic protrusion region of the enzyme. Such an interaction seems to require a conformational change in the enzyme or substrate, or in both. Determination of kinetic parameters and stoichiometry of RNase HI molecules bound to substrates of various lengths would provide additional information about the binding of enzyme to substrate.Magnesium ions may also modulate protein-nucleic acid ...

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Purification and characterization of the RNase H domain of HIV‐1 reverse transcriptase expressed in recombinant Escherichia coli

Cited by 55 publications

References 18 publications

Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

Mutations within the Primer Grip Region of HIV-1 Reverse Transcriptase Result in Loss of RNase H Function

Substitution of a highly basic helix/loop sequence into the RNase H domain of human immunodeficiency virus reverse transcriptase restores its Mn(2+)-dependent RNase H activity.

Kinetic and Stoichiometric Analysis for the Binding of Escherichia coli Ribonuclease HI to RNA-DNA Hybrids Using Surface Plasmon Resonance

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