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Wei Yang - One of the best experts on this subject based on the ideXlab platform.
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structure of hiv 1 reverse transcriptase cleaving rna in an rna DNA Hybrid
Proceedings of the National Academy of Sciences of the United States of America, 2018Co-Authors: Lan Tian, Min Sung Kim, Jimin Wang, Wei YangAbstract:HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-A resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA Hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA Hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 A. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.
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Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA Hybrid.
Proceedings of the National Academy of Sciences, 2018Co-Authors: Lan Tian, Min Sung Kim, Jimin Wang, Wei YangAbstract:HIV-1 reverse transcriptase (RT) contains both DNA polymerase and RNase H activities to convert the viral genomic RNA to dsDNA in infected host cells. Here we report the 2.65-A resolution structure of HIV-1 RT engaging in cleaving RNA in an RNA/DNA Hybrid. A preferred substrate sequence is absolutely required to enable the RNA/DNA Hybrid to adopt the distorted conformation needed to interact properly with the RNase H active site in RT. Substituting two nucleotides 4 bp upstream from the cleavage site results in scissile-phosphate displacement by 4 A. We also have determined the structure of HIV-1 RT complexed with an RNase H-resistant polypurine tract sequence, which adopts a rigid structure and is accommodated outside of the nuclease active site. Based on this newly gained structural information and a virtual drug screen, we have identified an inhibitor specific for the viral RNase H but not for its cellular homologs.
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Complexes of HIV-1 RT, NNRTI and RNA/DNA Hybrid reveal a structure compatible with RNA degradation
Nature Structural & Molecular Biology, 2013Co-Authors: Mikalai Lapkouski, Lan Tian, Jennifer T Miller, Stuart F J Le Grice, Wei YangAbstract:Crystal structures of HIV-1 reverse transcriptase (RT) bound to an RNA/DNA Hybrid (without any cross-linking) and in the presence of non-nucleotide RT inhibitors (NNRTIs) nevirapine and efavirenz are now reported. The structures show the RNA/DNA Hybrid in a previously unseen conformation with ready access to the RNase-H active site of RT. Hundreds of structures of type 1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined, but only one contains an RNA/DNA Hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA Hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid–RT structures including the RNA/DNA Hybrid. The enzyme structure also differs from all previous RT–DNA complexes. Thus, the Hybrid has ready access to the RNase-H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT-Hybrid interface that undergoes conformational changes between two catalytic states.
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complexes of hiv 1 rt nnrti and rna DNA Hybrid reveal a structure compatible with rna degradation
Nature Structural & Molecular Biology, 2013Co-Authors: Mikalai Lapkouski, Lan Tian, Jennifer T Miller, Stuart Le F J Grice, Wei YangAbstract:Crystal structures of HIV-1 reverse transcriptase (RT) bound to an RNA/DNA Hybrid (without any cross-linking) and in the presence of non-nucleotide RT inhibitors (NNRTIs) nevirapine and efavirenz are now reported. The structures show the RNA/DNA Hybrid in a previously unseen conformation with ready access to the RNase-H active site of RT.
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Complexes of HIV-1 RT, NNRTI and RNA/DNA Hybrid reveal a structure compatible with RNA degradation
Nature Structural & Molecular Biology, 2013Co-Authors: Mikalai Lapkouski, Lan Tian, Jennifer T Miller, Stuart F J Le Grice, Wei YangAbstract:Crystal structures of HIV-1 reverse transcriptase (RT) bound to an RNA/DNA Hybrid (without any cross-linking) and in the presence of non-nucleotide RT inhibitors (NNRTIs) nevirapine and efavirenz are now reported. The structures show the RNA/DNA Hybrid in a previously unseen conformation with ready access to the RNase-H active site of RT.
Brian Luke - One of the best experts on this subject based on the ideXlab platform.
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The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA Hybrid-mediated DNA damage.
PLOS Genetics, 2017Co-Authors: Juan Lafuente-barquero, Marco Graf, Sonia Silva, Arianna Lockhart, Michael Lisby, Andres Aguilera, Sarah Luke-glaser, Belén Gómez-gonzález, Brian LukeAbstract:RNA-DNA Hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA Hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA Hybrids and RNA-DNA Hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA Hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at Hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA Hybrids.
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the smc5 6 complex regulates the yeast mph1 helicase at rna DNA Hybrid mediated DNA damage
PLOS Genetics, 2017Co-Authors: Juan Lafuentebarquero, Sarah Lukeglaser, Marco Graf, Sonia Silva, Belen Gomezgonzalez, Arianna Lockhart, Michael Lisby, Andres Aguilera, Brian LukeAbstract:: RNA-DNA Hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA Hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA Hybrids and RNA-DNA Hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA Hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at Hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA Hybrids.
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The differential processing of telomeres in response to increased telomeric transcription and RNA–DNA Hybrid accumulation
RNA Biology, 2014Co-Authors: Bettina Balk, Martina Dees, Katharina Bender, Brian LukeAbstract:Telomeres are protective nucleoprotein structures at the ends of eukaryotic chromosomes. Despite the heterochromatic state of telomeres they are transcribed, generating non-coding telomeric repeat-containing RNA (TERRA). Strongly induced TERRA transcription has been shown to cause telomere shortening and accelerated senescence in the absence of both telomerase and homology-directed repair (HDR). Moreover, it has recently been demonstrated that TERRA forms RNA–DNA Hybrids at chromosome ends. The accumulation of RNA–DNA Hybrids at telomeres also leads to rapid senescence and telomere loss in the absence of telomerase and HDR. Conversely, in the presence of HDR, telomeric RNA–DNA Hybrid accumulation and increased telomere transcription promote telomere recombination, and hence, delayed senescence. Here, we demonstrate that despite these similar phenotypic outcomes, telomeres that are highly transcribed are not processed in the same manner as those that accumulate RNA–DNA Hybrids.
David A. Clayton - One of the best experts on this subject based on the ideXlab platform.
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properties of a primer rna DNA Hybrid at the mouse mitochondrial DNA leading strand origin of replication
Journal of Biological Chemistry, 1996Co-Authors: David A. ClaytonAbstract:Abstract Primers for vertebrate mitochondrial leading-strand DNA replication are products of transcription synthesized by mitochondrial RNA polymerase. The precursor primer RNA exists as a persistent RNA-DNA Hybrid, known as an R-loop, formed during transcription through the replication origin (Xu, B., and Clayton, D. A. (1996) EMBO J. 15, 3135-3143). In an effort to examine the precise structure of this primer RNA intermediate, we have used two methods to reconstitute model R-loops containing the mouse mitochondrial DNA origin sequence. First, we demonstrate that bacteriophage SP6 RNA polymerase can efficiently catalyze the formation of an R-loop at the mouse mtDNA origin sequence. Second, the R-loop can be assembled by annealing presynthesized RNA and supercoiled DNA template in the presence of formamide. R-loop formation by either method is dependent on specific template sequences. The reconstituted R-loop is exceptionally stable and exhibits an unexpected structure. Structural studies indicate that the RNA strand is organized within the RNA-DNA base-paired region, suggesting that the heteroduplex interaction occurs through a specific conformation. We propose that the organized structure of the R-loop is critical for primer RNA function in vivo with important implications for the RNA processing and DNA replication machinery.
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rna DNA Hybrid formation at the human mitochondrial heavy strand origin ceases at replication start sites an implication for rna DNA Hybrids serving as primers
The EMBO Journal, 1996Co-Authors: Baoji Xu, David A. ClaytonAbstract:Abstract Critical elements of a mammalian mitochondrial DNA heavy-strand replication origin include a promoter and three downstream conserved sequence blocks (CSBIII, CSBII and CSBI). We found recently that a stable and persistent RNA-DNA Hybrid forms during in vitro transcription at Saccharomyces cerevisiae mitochondrial origins; Hybrid formation was dependent on the conserved CSBII element. We report here that during in vitro transcription with human mitochondrial RNA polymerase, stable and persistent RNA-DNA Hybrid formation is also evident at the human mitochondrial heavy-strand origin. As predicted, Hybrid formation was dependent on the GC-rich CSBII element. The human RNA-DNA Hybrids terminate within or downstream of CSBI at locations implicated in initiation of mitochondrial DNA replication. Interestingly, efficient Hybrid formation in the human system is influenced by sequence 5' to the RNA-DNA Hybrid, including the CSBIII element. These results suggest that the RNA-DNA Hybrids formed during transcription across the mitochondrial DNA heavy-strand origin provide RNA primers for initiation of mitochondrial DNA replication.
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A Persistent RNA-DNA Hybrid Is Formed during Transcription at a Phylogenetically Conserved Mitochondrial DNA Sequence
Molecular and Cellular Biology, 1995Co-Authors: David A. ClaytonAbstract:Critical features of the mitochondrial leading-strand DNA replication origin are conserved from Saccharomyces cerevisiae to humans. These include a promoter and a downstream GC-rich sequence block (CSBII) that encodes rGs within the primer RNA. During in vitro transcription at yeast mitochondrial replication origins, there is stable and persistent RNA-DNA Hybrid formation that begins at the 5' end of the rG region. The short rG-dC sequence is the necessary and sufficient nucleic acid element for establishing stable Hybrids, and the presence of rGs within the RNA strand of the RNA-DNA Hybrid is required. The efficiency of Hybrid formation depends on the length of RNA synthesized 5' to CSBII and the type of RNA polymerase employed. Once made, the RNA strand of an RNA-DNA Hybrid can serve as an effective primer for mitochondrial DNA polymerase. These results reveal a new mechanism for persistent RNA-DNA Hybrid formation and suggest a step in priming mitochondrial DNA replication that requires both mitochondrial RNA polymerase and an rG-dC sequence-specific event to form an extensive RNA-DNA Hybrid.
Marcin Nowotny - One of the best experts on this subject based on the ideXlab platform.
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Ty3 reverse transcriptase complexed with an RNA-DNA Hybrid shows structural and functional asymmetry
Nature Structural & Molecular Biology, 2014Co-Authors: Elżbieta Nowak, Jennifer T Miller, Stuart F J Le Grice, Marion K. Bona, Justyna Studnicka, Roman H. Szczepanowski, Jakub Jurkowski, Marcin NowotnyAbstract:A new study reports the first structure of a retrotransposon reverse transcriptase in complex with its cognate polypurine tract RNA-DNA Hybrid. In contrast to its retroviral counterparts, Ty3 reverse transcriptase forms an asymmetric homodimer that forms in the presence of substrate, with its RNase H and DNA polymerase activities likely contributed by separate subunits. Retrotransposons are a class of mobile genetic elements that replicate by converting their single-stranded RNA intermediate to double-stranded DNA through the combined DNA polymerase and ribonuclease H (RNase H) activities of the element-encoded reverse transcriptase (RT). Although a wealth of structural information is available for lentiviral and gammaretroviral RTs, equivalent studies on counterpart enzymes of long terminal repeat (LTR)–containing retrotransposons, from which they are evolutionarily derived, is lacking. In this study, we report the first crystal structure of a complex of RT from the Saccharomyces cerevisiae LTR retrotransposon Ty3 in the presence of its polypurine tract–containing RNA-DNA Hybrid. In contrast to its retroviral counterparts, Ty3 RT adopts an asymmetric homodimeric architecture whose assembly is substrate dependent. Moreover, our structure and biochemical data suggest that the RNase H and DNA polymerase activities are contributed by individual subunits of the homodimer.
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Ty3 reverse transcriptase complexed with an RNA-DNA Hybrid shows structural and functional asymmetry
Nature Structural & Molecular Biology, 2014Co-Authors: Elzbieta Nowak, Jennifer T Miller, Stuart F J Le Grice, Marion K. Bona, Justyna Studnicka, Roman H. Szczepanowski, Jakub Jurkowski, Marcin NowotnyAbstract:A new study reports the first structure of a retrotransposon reverse transcriptase in complex with its cognate polypurine tract RNA-DNA Hybrid. In contrast to its retroviral counterparts, Ty3 reverse transcriptase forms an asymmetric homodimer that forms in the presence of substrate, with its RNase H and DNA polymerase activities likely contributed by separate subunits.
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Specific recognition of RNA/DNA Hybrid and enhancement of human RNase H1 activity by HBD.
The EMBO Journal, 2008Co-Authors: Marcin Nowotny, Susana M. Cerritelli, Rodolfo Ghirlando, Sergei Gaidamakov, Robert J. Crouch, Wei YangAbstract:Human RNase H1 contains an N-terminal domain known as dsRHbd for binding both dsRNA and RNA/DNA Hybrid. We find that dsRHbd binds preferentially to RNA/DNA Hybrids by over 25-fold and rename it as Hybrid binding domain (HBD). The crystal structure of HBD complexed with a 12 bp RNA/DNA Hybrid reveals that the RNA strand is recognized by a protein loop, which forms hydrogen bonds with the 2′-OH groups. The DNA interface is highly specific and contains polar residues that interact with the phosphate groups and an aromatic patch that appears selective for binding deoxyriboses. HBD is unique relative to non-sequence-specific dsDNA- and dsRNA-binding domains because it does not use positive dipoles of α-helices for nucleic acid binding. Characterization of full-length enzymes with defective HBDs indicates that this domain dramatically enhances both the specific activity and processivity of RNase H1. Similar activity enhancement by small substrate-binding domains linked to the catalytic domain likely occurs in other nucleic acid enzymes.
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crystal structures of rnase h bound to an rna DNA Hybrid substrate specificity and metal dependent catalysis
Cell, 2005Co-Authors: Marcin Nowotny, Sergei Gaidamakov, Robert J. Crouch, Wei YangAbstract:RNase H belongs to a nucleotidyl-transferase superfamily, which includes transposase, retroviral integrase, Holliday junction resolvase, and RISC nuclease Argonaute. We report the crystal structures of RNase H complexed with an RNA/DNA Hybrid and a mechanism for substrate recognition and two-metal-ion-dependent catalysis. RNase H specifically recognizes the A form RNA strand and the B form DNA strand. Structure comparisons lead us to predict the catalytic residues of Argonaute and conclude that two-metal-ion catalysis is a general feature of the superfamily. In nucleases, the two metal ions are asymmetrically coordinated and have distinct roles in activating the nucleophile and stabilizing the transition state. In transposases, they are symmetrically coordinated and exchange roles to alternately activate a water and a 3'-OH for successive strand cleavage and transfer by a ping-pong mechanism.
Sonia Silva - One of the best experts on this subject based on the ideXlab platform.
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the smc5 6 complex regulates the yeast mph1 helicase at rna DNA Hybrid mediated DNA damage
PLOS Genetics, 2017Co-Authors: Juan Lafuentebarquero, Sarah Lukeglaser, Marco Graf, Sonia Silva, Belen Gomezgonzalez, Arianna Lockhart, Michael Lisby, Andres Aguilera, Brian LukeAbstract:: RNA-DNA Hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA Hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA Hybrids and RNA-DNA Hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA Hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at Hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1's helicase activity plays a crucial role in responding to persistent RNA-DNA Hybrids.
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The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA Hybrid-mediated DNA damage.
PLOS Genetics, 2017Co-Authors: Juan Lafuente-barquero, Marco Graf, Sonia Silva, Arianna Lockhart, Michael Lisby, Andres Aguilera, Sarah Luke-glaser, Belén Gómez-gonzález, Brian LukeAbstract:RNA-DNA Hybrids are naturally occurring obstacles that must be overcome by the DNA replication machinery. In the absence of RNase H enzymes, RNA-DNA Hybrids accumulate, resulting in replication stress, DNA damage and compromised genomic integrity. We demonstrate that Mph1, the yeast homolog of Fanconi anemia protein M (FANCM), is required for cell viability in the absence of RNase H enzymes. The integrity of the Mph1 helicase domain is crucial to prevent the accumulation of RNA-DNA Hybrids and RNA-DNA Hybrid-dependent DNA damage, as determined by Rad52 foci. Mph1 forms foci when RNA-DNA Hybrids accumulate, e.g. in RNase H or THO-complex mutants and at short telomeres. Mph1, however is a double-edged sword, whose action at Hybrids must be regulated by the Smc5/6 complex. This is underlined by the observation that simultaneous inactivation of RNase H2 and Smc5/6 results in Mph1-dependent synthetic lethality, which is likely due to an accumulation of toxic recombination intermediates. The data presented here support a model, where Mph1’s helicase activity plays a crucial role in responding to persistent RNA-DNA Hybrids.