Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals

Götte, Matthias; Maier, Gottfried; Groß, H.; Heumann, Hermann

doi:10.1074/jbc.273.17.10139

Cited by 52 publications

(65 citation statements)

References 36 publications

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“…We have previously applied high resolution footprinting techniques to study the interaction between HIV-1 RT and its DNA/DNA primer/template substrate (27,28). Two chemically distinct sources of hydroxyl radicals were utilized: the classical method with [Fe(EDTA)] 2Ϫ that yields hydroxyl radicals via Fenton-like chemistry; and potassium peroxynitrite (KOONO), which is a metal-free source of hydroxyl radicals that are generated during decomposition of the conjugate acid HOONO.…”

Section: Resultsmentioning

confidence: 99%

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Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Marchand

Götte

2003

Journal of Biological Chemistry

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146

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Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre-and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

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Section: Resultsmentioning

confidence: 99%

“…Prior to the treatment with KOONO or Fe 2ϩ , complexes were preincubated for 20 min with increasing concentrations of the next nucleotide and at different temperatures as indicated in the figures. Treatment with Fe 2ϩ and KOONO, respectively, was performed essentially as described recently (26,27).…”

Section: Methodsmentioning

confidence: 99%

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Marchand

Götte

2003

Journal of Biological Chemistry

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146

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“…This mode of binding places the RNase H active site 16-19 nt downstream on the RNA, where a primary cut is made (20). The length of the product of this cleavage corresponds to the distance between the polymerase and RNase H active sites as shown by crystallographic, biochemical, and footprinting data (23)(24)(25)(26)(27). RT also can move or position the polymerase active site further 3Ј on the DNA template to place the RNase H site 8-9 nt from the 5Ј end of the RNA to create the secondary cut.…”

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confidence: 99%

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Wisniewski

Balakrishnan

Palaniappan

et al. 2000

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

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HIV-1 reverse transcriptase (RT) degrades the plus strand viral RNA genome while synthesizing the minus strand of DNA. Many RNA fragments, including the polypurine tracts, remain annealed to the new DNA. Several RTs are believed to bind after synthesis to degrade all RNA fragments except the polypurine tracts by a polymerization-independent mode of RNase H activity. For this latter process, we found that RT positions the RNase H active site approximately 18 nt from the 5 end of the RNA, making the primary cut. The enzyme rebinds or slides toward the 5 end of the RNA to make a secondary cut creating two products 8 -9 nt long. RT then binds the new 5 end of the RNA created by the first primary or the secondary cuts to make the next primary cut. In addition, we observed another type of RNase H cleavage specificity. RT aligns the RNase H active site to the 3 end of the RNA, cutting 5 residues in. We determined the relative rates of these cuts, defining their temporal order. Results show that the first primary cut is fastest, and the secondary and 5-nt cuts occur next at similar rates. The second primary cuts appear last. Based on these results, we present a model by which RT progressively cleaves RNA fragments.H IV is the causative agent of AIDS (1). To establish an infection, HIV must integrate a double-stranded viral DNA into the host chromosome (2). The virus-encoded reverse transcriptase (RT) converts the single-stranded viral RNA genome into that double-stranded DNA. RT uses several different catalytic activities in this process. The polymerase function catalyzes DNA synthesis on RNA and DNA templates (3). RT also has an RNase H active site that cleaves RNA when annealed to DNA. RNase H activity is required for a number of steps in viral replication, including formation of primers and primer strand transfers (4). An important role for the RNase H is the complete removal of the original genomic RNA during the synthesis of the double-stranded DNA (5, 6).The genomic viral RNA first is converted to an RNA͞DNA hybrid and then to double-stranded DNA. Synthesis of the second DNA strand necessitates complete removal of the original RNA (7,8). The RNA genome is cut into small segments during and after synthesis of the RNA͞DNA hybrid (9-11). Studies show that two different modes of RNase H activity are important for the removal of the RNA (5, 12, 13). The first mode is carried out by the same RT performing DNA synthesis. This is the polymerization-dependent mode of cleavage (9)(10)(11)(14)(15)(16)(17). We showed that the HIV-1 RT synthesizing DNA leaves RNA oligomers in its wake, many still bound to the extended DNA primer (17, 18). These residual RNAs occur because cleavages of the RNA template happen less frequently than nucleotide addition. Kati et al. (19) examined the rates of both catalytic activities of RT and found that the polymerization rate is 7-10 times faster than the RNase H rate. This result demonstrates that the two activities are uncoupled, such that several nucleotides are added in the time required fo...

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“…Site-specific footprinting is a powerful technique for measuring the position of HIV-1 RT on a substrate to single-nucleotide resolution. RT⅐substrate complexes are treated with KOONO, which produces hydroxyl radicals and results in the hyper-reactive cleavage of the DNA substrate via Cys-280 at positions Ϫ7/Ϫ8, representing the post-and pretranslocational positions of RT, respectively (22,31). A cleavage at either of these positions indicates that RT is positioned at the 3Ј primer terminus (i.e.…”

Section: Resultsmentioning

confidence: 99%

“…The reaction was incubated at 37°C for 10 min after the addition of ligand (and another 10 min after the addition of dNTPs). Treatment with potassium peroxynitrite (KOONO) was performed essentially as described (22).…”

Section: Methodsmentioning

confidence: 99%

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

Auger

Beilhartz

Zhu

et al. 2011

Journal of Biological Chemistry

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The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre-and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA⅐DNA primer/template substrates versus DNA⅐DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA⅐DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.HIV RT is a multifunctional enzyme that catalyzes the conversion of the single-stranded RNA HIV genome to doublestranded DNA that is then incorporated into the host genome by the HIV integrase. In the process, RT must support RNAdirected and DNA-directed DNA synthesis along with ribonuclease H (RNase H) 2 activities to create an integration-competent product. A key step during reverse transcription is the incomplete hydrolysis of the (ϩ)-stranded RNA that leaves behind a purine-rich RNA sequence referred to as the polypurine tract (PPT). The remnant RNA PPT sequence serves as a primer to initiate synthesis of the (ϩ)-strand DNA (second strand) (1-3). The unique structure of the RNA PPT sequence has been shown to be a key determinant of the RNase H cleavage specificity at the PPT/U3 junction (4). A recent report described the mechanism by which RT discriminates between polymerase and RNase H activities thereby enabling more efficient initiation of polymerization from the RNA PPT primer versus other remnant RNA primers (5). Using single-molecule spectroscopy experiments, it was shown that RT binds nucleic acid substrates in two distinct orientations in a manner that is governed by the sugar backbone composition of the four or five nucleotides at each end of the primer. Depending on the binding orientation, RT either initiates polymerization at the 3Ј-end of the primer (polymerase binding mode on a DNA primer), or alternatively, RNA hydrolysis through the RNase H domain (RNase H bindi...

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Localization of the Active Site of HIV-1 Reverse Transcriptase-associated RNase H Domain on a DNA Template Using Site-specific Generated Hydroxyl Radicals

Cited by 52 publications

References 36 publications

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

Unique progressive cleavage mechanism of HIV reverse transcriptase RNase H

Impact of Primer-induced Conformational Dynamics of HIV-1 Reverse Transcriptase on Polymerase Translocation and Inhibition

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