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Edward Arnold - One of the best experts on this subject based on the ideXlab platform.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:Abstract The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3′ terminus of the primer strand. Observation of a hydrogen bond between the 3′-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the “primer grip” (the β12-β13 hairpin) with the 3′ terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable π-π interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices αH and αI of the p66 thumb) can be thought of functioning as a “translocation track” that guides the relative movement of nucleic acid and protein during polymerization.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Kalyan Das, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.
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protein nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double stranded DNA Template primer
Biopolymers, 1997Co-Authors: Jianping Ding, Stephen H. Hughes, Edward ArnoldAbstract:The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "Template grip" have close contacts with the Template strand and aid in positioning the Template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the Template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
C Richardson - One of the best experts on this subject based on the ideXlab platform.
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zinc binding domain of the bacteriophage t7 DNA primase modulates binding to the DNA Template
Journal of Biological Chemistry, 2012Co-Authors: Barak Akabayov, C RichardsonAbstract:The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA Template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze Template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short Templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis.
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a covalent linkage between the gene 5 DNA polymerase of bacteriophage t7 and escherichia coli thioredoxin the processivity factor fate of thioredoxin during DNA synthesis
Journal of Biological Chemistry, 2003Co-Authors: Donald E Johnson, C RichardsonAbstract:Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-Template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA Template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA Template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.
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modular architecture of the bacteriophage t7 primase couples rna primer synthesis to DNA synthesis
Molecular Cell, 2003Co-Authors: Masato Kato, Gerhard Wagner, C Richardson, Tom EllenbergerAbstract:Abstract DNA primases are Template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg 2+ ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA Template in the primase active site and then delivering the primed DNA Template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.
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interaction of bacteriophage t7 gene 4 primase with its Template recognition site
Journal of Biological Chemistry, 1999Co-Authors: David N Frick, C RichardsonAbstract:Abstract The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA Template containing a primase recognition site (5′-GGGTC-3′, 5′-TGGTC-3′, 5′-GTGTC-3′, or 5′-TTGTC-3′). Although it is not copied into the oligoribonucleotides, the cytosine at the 3′-position is essential for synthesis and Template binding. Two nucleotides flanking the 3′-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5′-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA Template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for Templates is weak, ranging from 10 to 150 μm. The tight DNA binding (<1 μm) observed with the 63-kDa gene 4 protein occurs viainteractions between DNA Templates and the helicase domain.
Jianping Ding - One of the best experts on this subject based on the ideXlab platform.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:Abstract The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3′ terminus of the primer strand. Observation of a hydrogen bond between the 3′-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the “primer grip” (the β12-β13 hairpin) with the 3′ terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable π-π interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices αH and αI of the p66 thumb) can be thought of functioning as a “translocation track” that guides the relative movement of nucleic acid and protein during polymerization.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Kalyan Das, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.
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protein nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double stranded DNA Template primer
Biopolymers, 1997Co-Authors: Jianping Ding, Stephen H. Hughes, Edward ArnoldAbstract:The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "Template grip" have close contacts with the Template strand and aid in positioning the Template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the Template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
Stephen H. Hughes - One of the best experts on this subject based on the ideXlab platform.
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HIV-1 reverse transcriptase connection subdomain mutations reduce Template RNA degradation and enhance AZT excision
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Krista A. Delviks-frankenberry, Galina N. Nikolenko, Paul L. Boyer, Stephen H. Hughes, John M. Coffin, Abhay Jere, Vinay K. PathakAbstract:We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and Template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA Template compared with a DNA Template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA Template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing Template RNA degradation, thereby providing additional time for RT to excise AZTMP.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Kalyan Das, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3' terminus of the primer strand. Observation of a hydrogen bond between the 3'-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the "primer grip" (the beta12-beta13 hairpin) with the 3' terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable pi-pi interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices alphaH and alphaI of the p66 thumb) can be thought of functioning as a "translocation track" that guides the relative movement of nucleic acid and protein during polymerization.
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structure and functional implications of the polymerase active site region in a complex of hiv 1 rt with a double stranded DNA Template primer and an antibody fab fragment at 2 8 a resolution
Journal of Molecular Biology, 1998Co-Authors: Jianping Ding, Stephen H. Hughes, Y Hsiou, Stefan G Sarafianos, Arthur D Clark, Alfredo Jacobomolina, Chris Tantillo, Edward ArnoldAbstract:Abstract The structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) complexed with a 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) and the Fab fragment of monoclonal antibody 28 (Fab28) has been refined at 2.8 A resolution. The structures of the polymerase active site and neighboring regions are described in detail and a number of novel insights into mechanisms of polymerase catalysis and drug inhibition are presented. The three catalytically essential amino acid residues (Asp110, Asp185, and Asp186) are located close to the 3′ terminus of the primer strand. Observation of a hydrogen bond between the 3′-OH of the primer terminus and the side-chain of Asp185 suggests that the carboxylate of Asp185 could act as a general base in initiating the nucleophilic attack during polymerization. Nearly all of the close protein-DNA interactions involve atoms of the sugar-phosphate backbone of the nucleic acid. However, the phenoxyl side-chain of Tyr183, which is part of the conserved YMDD motif, has hydrogen-bonding interactions with nucleotide bases of the second duplex base-pair and is predicted to have at least one hydrogen bond with all Watson-Crick base-pairs at this position. Comparison of the structure of the active site region in the HIV-1 RT/dsDNA complex with all other HIV-1 RT structures suggests that Template-primer binding is accompanied by significant conformational changes of the YMDD motif that may be relevant for mechanisms of both polymerization and inhibition by non-nucleoside inhibitors. Interactions of the “primer grip” (the β12-β13 hairpin) with the 3′ terminus of the primer strand primarily involve the main-chain atoms of Met230 and Gly231 and the primer terminal phosphate. Alternative positions of the primer grip observed in different HIV-1 RT structures may be related to conformational changes that normally occur during DNA polymerization and translocation. In the vicinity of the polymerase active site, there are a number of aromatic residues that are involved in energetically favorable π-π interactions and may be involved in the transitions between different stages of the catalytic process. The protein structural elements primarily responsible for precise positioning of the Template-primer (including the primer grip, Template grip, and helices αH and αI of the p66 thumb) can be thought of functioning as a “translocation track” that guides the relative movement of nucleic acid and protein during polymerization.
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protein nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double stranded DNA Template primer
Biopolymers, 1997Co-Authors: Jianping Ding, Stephen H. Hughes, Edward ArnoldAbstract:The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA Template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "Template grip" have close contacts with the Template strand and aid in positioning the Template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the Template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
Tom Ellenberger - One of the best experts on this subject based on the ideXlab platform.
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a molecular handoff between bacteriophage t7 DNA primase and t7 DNA polymerase initiates DNA synthesis
Journal of Biological Chemistry, 2004Co-Authors: Masato Kato, Gerhard Wagner, Tom EllenbergerAbstract:Abstract The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA Template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA Template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed Template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.
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modular architecture of the bacteriophage t7 primase couples rna primer synthesis to DNA synthesis
Molecular Cell, 2003Co-Authors: Masato Kato, Gerhard Wagner, C Richardson, Tom EllenbergerAbstract:Abstract DNA primases are Template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg 2+ ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA Template in the primase active site and then delivering the primed DNA Template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.