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Millie M. Georgiadis - One of the best experts on this subject based on the ideXlab platform.
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THE CRYSTAL STRUCTURE OF THE MONOMERIC REVERSE TRANSCRIPTASE FROM MOLONEY MURINE LEUKEMIA VIRUS
Structure (London England : 1993), 2004Co-Authors: Debanu Das, Millie M. GeorgiadisAbstract:Reverse transcriptases (RTs) are multidomain enzymes of variable architecture that couple both RNA- and DNA-directed DNA Polymerase activities with an RNase H activity specific for an RNA:DNA hybrid in order to replicate the single-stranded RNA genome of the retrovirus. Previous structural work has been reported for the heterodimeric HIV-1 and HIV-2 RTs. We now report the first crystal structure of the full-length Moloney murine leukemia virus (MMLV) RT at 3.0 A resolution. The structure reveals a clamp-shaped molecule resulting from the relative positions of the thumb, connection, and RNase H domains that is strikingly different from the HIV-1 RT and provides the first example of a monomeric reverse transcriptase. A comparative analysis with related DNA Polymerases suggests a unique trajectory for the template-primer exiting the Polymerase active site and provides insights regarding processive DNA synthesis by MMLV RT.
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Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain
Journal of molecular biology, 2000Co-Authors: Shabir Najmudin, Dunming Sun, Marie L. Coté, Sarah J. Yohannan, Sherwin P. Montano, Millie M. GeorgiadisAbstract:Reverse transcriptase (RT) serves as the replicative Polymerase for retroviruses by using RNA and DNA-directed DNA Polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.
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Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Protein science : a publication of the Protein Society, 1998Co-Authors: Dunming Sun, Sven M Jessen, Chunhui Liu, Xiuping Liu, Shabir Najmudin, Millie M. GeorgiadisAbstract:Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA Polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the Polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain Polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
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Mechanistic implications from the structure of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase
Structure (London England : 1993), 1995Co-Authors: Millie M. Georgiadis, Sven M Jessen, Craig M. Ogata, Alice Telesnitsky, Stephen P. Goff, Wayne A. HendricksonAbstract:Abstract Background: Reverse transcriptase (RT) converts the single-stranded RNA genome of a retrovirus into a double-stranded DNA copy for integration into the host genome. This process requires ribonuclease H as well as RNA- and DNA-directed DNA Polymerase activities. Although the overall organization of HIV-1 RT is known from previously reported crystal structures, no structure of a complex including a metal ion, which is essential for its catalytic activity, has been reported. Results Here we describe the structures at 1.8 a resolution of a catalytically active fragment of RT from Moloney murine leukemia virus (MMLV) and at 2.6 a of a complex of this fragment with Mn 2+ coordinated in the Polymerase active site. On the basis of similarities with HIV-1 RT and rat DNA Polymerase β , we have modeled template/primer and deoxyribonucleoside 5′-triphosphate substrates into the MMLV RT structure. Conclusion Our model, in the context of the disposition of evolutionarily conserved residues seen here at high resolution, provides new insights into the mechanisms of catalysis, fidelity, processivity and discrimination between deoxyribose and ribose nucleotides.
Luis Blanco - One of the best experts on this subject based on the ideXlab platform.
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Altered hematopoiesis in mice lacking DNA Polymerase μ is due to inefficient double-strand break repair
PLoS genetics, 2009Co-Authors: Daniel Lucas, Beatriz Escudero, José M. Ligos, José C. Segovia, Juan Camilo Estrada, Gloria Terrados, Luis Blanco, Enrique Samper, Antonio BernadAbstract:Polymerase mu (Polμ) is an error-prone, DNA-directed DNA Polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polμ deficiency results in impaired Vκ-Jκ recombination and altered somatic hypermutation and centroblast development. In Polμ−/− mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body γ-irradiation revealed that Polμ also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polμ function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.
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DNA Polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA.
Journal of molecular biology, 2003Co-Authors: Ramón García-escudero, Luis Blanco, María L. Salas, Miguel Garcia-diaz, José SalasAbstract:Abstract DNA Polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA Polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3′–5′ exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1 nt gapped DNA; (iii) a 5′-phosphorylated 1 nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5′-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10 −4 to 10 −5 , and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1 nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5′-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.
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Characterization of an African Swine Fever Virus 20-kDa DNA Polymerase Involved in DNA Repair
The Journal of biological chemistry, 1997Co-Authors: Mariano Oliveros, Rafael J. Yáñez, María L. Salas, José Salas, Eladio Viñuela, Luis BlancoAbstract:Abstract African swine fever virus (ASFV) encodes a novel DNA Polymerase, constituted of only 174 amino acids, belonging to the Polymerase (pol) X family of DNA Polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA Polymerase, highly distributive, lacking a proofreading 3′-5′-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA Polymerases, together with the extrapolation to the crystal structure of mammalian DNA Polymerase β (pol β), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol β-type DNA Polymerase core, constituted by only the “palm” and “thumb” subdomains. It is worth noting that such an “unfingered” DNA Polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.
Shabir Najmudin - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain
Journal of molecular biology, 2000Co-Authors: Shabir Najmudin, Dunming Sun, Marie L. Coté, Sarah J. Yohannan, Sherwin P. Montano, Millie M. GeorgiadisAbstract:Reverse transcriptase (RT) serves as the replicative Polymerase for retroviruses by using RNA and DNA-directed DNA Polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.
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Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Protein science : a publication of the Protein Society, 1998Co-Authors: Dunming Sun, Sven M Jessen, Chunhui Liu, Xiuping Liu, Shabir Najmudin, Millie M. GeorgiadisAbstract:Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA Polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the Polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain Polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
Thomas H. Eickbush - One of the best experts on this subject based on the ideXlab platform.
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DNA-directed DNA Polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon.
Journal of molecular biology, 2007Co-Authors: Anna Kurzynska-kokorniak, Varuni K. Jamburuthugoda, Arkadiusz Bibillo, Thomas H. EickbushAbstract:R2 elements are non-long terminal repeat (non-LTR) retrotransposons with a single open reading-frame encoding reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The elements are specialized for insertion into the 28 S rRNA genes of many animal phyla. The R2-encoded activities initiate retrotransposition by sequence-specific cleavage of the 28 S gene target site and the utilization of the released DNA 3' end to prime reverse transcription (target primed reverse transcription). The activity of the R2 Polymerase on RNA templates has been shown to differ from retroviral reverse transcriptases (RTs) in a number of properties. We demonstrate that the R2-RT is capable of efficiently utilizing single-stranded DNA (ssDNA) as a template. The processivity of the enzyme on ssDNA templates is higher than its processivity on RNA templates. This finding suggests that R2-RT is also capable of synthesizing the second DNA strand during retrotransposition. However, R2-RT lacks the RNAse H activity that is typically used by retroviral and LTR-retrotransposon RTs to remove the RNA strand before the first DNA strand is used as template. Remarkably, R2-RT can displace RNA strands that are annealed to ssDNA templates with essentially no loss of processivity. Such strand displacement activity is highly unusual for a DNA Polymerase. Thus the single R2 protein contains all the activities needed to make a double-stranded DNA product from an RNA transcript. Finally, during these studies we found an unexpected property of the highly sequence-specific R2 endonuclease domain. The endonuclease can non-specifically cleave ssDNA at a junction with double-stranded DNA. This activity suggests that second-strand cleavage of the target site may not be sequence specific, but rather is specified by a single-stranded region generated when the first DNA strand is used to prime reverse transcription.
Dunming Sun - One of the best experts on this subject based on the ideXlab platform.
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Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain
Journal of molecular biology, 2000Co-Authors: Shabir Najmudin, Dunming Sun, Marie L. Coté, Sarah J. Yohannan, Sherwin P. Montano, Millie M. GeorgiadisAbstract:Reverse transcriptase (RT) serves as the replicative Polymerase for retroviruses by using RNA and DNA-directed DNA Polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.
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Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: Crystallization of nucleic acid complexes
Protein science : a publication of the Protein Society, 1998Co-Authors: Dunming Sun, Sven M Jessen, Chunhui Liu, Xiuping Liu, Shabir Najmudin, Millie M. GeorgiadisAbstract:Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA Polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the Polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain Polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.