ABSTRACT
TDP (tyrosyl-DNA phosphodiesterase) 1 catalyses the hydrolysis of phosphodiester linkages between a DNA 3' phosphate and a tyrosine residue as well as a variety of other DNA 3' substituents, and has been implicated in the repair of covalent complexes involving eukaryotic type IB topoisomerases. To better understand the substrate features that are recognized by TDP1, the size of either the DNA or protein component of the substrate was varied. Competition experiments and gel-shift analyses comparing a series of substrates with DNA lengths increasing from 6 to 28 nt indicated that, contrary to predictions based on the crystal structure of the protein, the apparent affinity for the substrate increased as the DNA length was increased over the entire range tested. It has been found previously that a substrate containing the full-length native form of human topoisomerase I protein is not cleaved by TDP1. Protein-oligonucleotide complexes containing either a 53 or 108 amino acid topoisomerase I-derived peptide were efficiently cleaved by TDP1, but similar to the full-length protein, a substrate containing a 333 amino acid topoisomerase I fragment was resistant to cleavage. Consistent with these results, evidence is presented that processing by the proteasome is required for TDP1 cleavage in vivo.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Phosphoric Diester Hydrolases/metabolism , Humans , Peptide Fragments/metabolism , Substrate SpecificityABSTRACT
The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design.
Subject(s)
HIV-1/enzymology , Moloney murine leukemia virus/enzymology , Nucleotides/genetics , Ribonuclease H/metabolism , Amino Acid Substitution/genetics , Base Sequence , DNA/genetics , Molecular Sequence Data , Protein Structure, Tertiary , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/chemistry , Ribonuclease H/isolation & purificationABSTRACT
To assay the preferential binding of eukaryotic type IB topoisomerases to supercoiled DNA, two methods are described that make use of a catalytically inactive mutant form of the enzyme. In the gel shift assay, the preference for binding to supercoiled plasmid DNA is detected in the presence of linear and nicked forms of the same DNA by a reduction in the mobility of the supercoiled plasmid during electrophoresis in agarose. The more quantitative filter binding assay compares the ability of nicked and supercoiled forms of the circular DNA to compete for the binding of a (3)H-labeled nicked DNA to the topoisomerase where the enzyme-DNA complexes are quantitated by the retention of the labeled DNA on a nitrocellulose membrane.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA, Superhelical/chemistry , DNA, Superhelical/metabolism , Electrophoretic Mobility Shift Assay/methods , Nucleic Acid Conformation , Binding, Competitive , DNA Breaks, Single-Stranded , DNA Topoisomerases, Type I/genetics , DNA, Superhelical/genetics , Humans , Plasmids/chemistry , Plasmids/genetics , Plasmids/metabolism , Protein BindingABSTRACT
The RNase H activity of reverse transcriptase carries out three types of cleavage termed internal, RNA 5' end-directed, and DNA 3' end-directed. Given the strong association between the polymerase domain of reverse transcriptase and a DNA 3' primer terminus, we asked whether the distance from the primer terminus is paramount for positioning DNA 3' end-directed cleavages or whether preferred sequences and/or a cleavage window are important as they are for RNA 5' end-directed cleavages. Using the reverse transcriptases of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV), we determined the effects of sequence, distance, and substrate end structure on DNA 3' end-directed cleavages. Utilizing sequence-matched substrates, our analyses showed that DNA 3' end-directed cleavages share the same sequence preferences as RNA 5' end-directed cleavages, but the sites must fall in a narrow window between the 15th and 20th nucleotides from the recessed end for HIV-1 reverse transcriptase and between the 17th and 20th nucleotides for M-MuLV. Substrates with an RNA 5' end recessed by 1 (HIV-1) or 2-3 (M-MuLV) bases on a longer DNA could accommodate both types of end-directed cleavage, but further recession of the RNA 5' end excluded DNA 3' end-directed cleavages. For HIV-1 RNase H, the inclusion of the cognate dNTP enhanced DNA 3' end-directed cleavages at the 17th and 18th nucleotides. These data demonstrate that all three modes of retroviral RNase H cleavage share sequence determinants that may be useful in designing assays to identify inhibitors of retroviral RNases H.
Subject(s)
HIV-1/genetics , Moloney murine leukemia virus/genetics , Retroviridae/genetics , Ribonuclease H/chemistry , Base Sequence , Catalytic Domain , Crystallography, X-Ray/methods , DNA/genetics , DNA, Viral/genetics , Humans , Molecular Sequence Data , Nucleic Acid Hybridization , Protein Structure, Tertiary , RNA, Viral/genetics , Retroviridae/enzymologyABSTRACT
Human topoisomerase I binds DNA in a topology-dependent fashion with a strong preference for supercoiled DNAs of either sign over relaxed circular DNA. One hypothesis to account for this preference is that a second DNA-binding site exists on the enzyme that mediates an association with the nodes present in supercoiled DNA. The failure of the enzyme to dimerize, even in the presence of DNA, appears to rule out the hypothesis that two binding sites are generated by dimerization of the protein. A series of mutant protein constructs was generated to test the hypotheses that the homeodomain-like core subdomain II (residues 233-319) provides a second DNA-binding site, or that the linker or basic residues in core subdomain III are involved in the preferential binding to supercoiled DNAs. When putative DNA contact points within core subdomain II were altered or the domain was removed altogether, there was no effect on the ability of the enzyme to recognize supercoiled DNA, as measured by both a gel shift assay and a competition binding assay. However, the preference for supercoils was noticeably reduced for a form of the enzyme lacking the coiled-coil linker region or when pairs of lysines were changed to glutamic acids in core subdomain III. The results obtained implicate the linker and solvent-exposed basic residues in core subdomain III in the preferential binding of human topoisomerase I to supercoiled DNA.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA, Superhelical/metabolism , DNA Mutational Analysis , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/genetics , DNA, Superhelical/chemistry , Electrophoretic Mobility Shift Assay , Humans , Models, Molecular , Mutation , Nucleic Acid Conformation , Protein Binding/genetics , Protein Binding/physiology , Protein Multimerization , Protein Structure, SecondaryABSTRACT
Retroviral reverse transcriptases possess both a DNA polymerase and an RNase H activity. The linkage with the DNA polymerase activity endows the retroviral RNases H with unique properties not found in the cellular counterparts. In addition to the typical endonuclease activity on a DNA/RNA hybrid, cleavage by the retroviral enzymes is also directed by both DNA 3' recessed and RNA 5' recessed ends, and by certain nucleotide sequence preferences in the vicinity of the cleavage site. This spectrum of specificities enables retroviral RNases H to carry out a series of cleavage reactions during reverse transcription that degrade the viral RNA genome after minus-strand synthesis, precisely generate the primer for the initiation of plus strands, facilitate the initiation of plus-strand synthesis and remove both plus- and minus-strand primers after they have been extended.
Subject(s)
Retroviridae/enzymology , Ribonuclease H/metabolism , Transcription, Genetic , Biocatalysis , Models, Molecular , Protein Conformation , Ribonuclease H/chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Human topoisomerase IB (hTopo) forms a covalent phosphotyrosyl linkage with the DNA backbone, and controls genomic DNA topology by relaxing DNA supercoils during the processes of DNA replication, transcription, chromosome condensation and decondensation. The essential role of hTopo in these processes has made it a preeminent anticancer drug target. We have screened a small library of arylstibonic acids for their effects on plasmid supercoil relaxation catalyzed by hTopo. Despite the similar structures of the library compounds, some compounds were found to be effective competitive inhibitors, and others, nonessential activators. Some arylstibonic acids show selectivity in their action against hTopo and the related enzyme from poxvirus (vTopo). Structure-activity relationships and structural modeling suggest that competitive inhibition may result from positioning of the negatively charged stibonic acid and carboxylate groups of the inhibitors into DNA phosphate binding pockets on hTopo. The hTopo activators act by a surprising allosteric mechanism without interfering with DNA binding or binding of the widely used hTopo poison camptothecin. Arylstibonic acid competitive inhibitors may become useful small molecules for elucidating the cellular functions of hTopo.
Subject(s)
Acids/chemistry , Acids/pharmacology , DNA Topoisomerases, Type I/metabolism , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Topoisomerase I Inhibitors , Camptothecin/chemistry , Camptothecin/pharmacology , DNA/metabolism , Enzyme Activation/drug effects , Humans , Molecular Structure , Protein Binding , Structure-Activity RelationshipABSTRACT
This review compares the well-studied RNase H activities of human immunodeficiency virus, type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) reverse transcriptases. The RNase H domains of HIV-1 and MoMLV are structurally very similar, with functions assigned to conserved subregions like the RNase H primer grip and the connection subdomain, as well as to distinct features like the C-helix and loop in MoMLV RNase H. Like cellular RNases H, catalysis by the retroviral enzymes appears to involve a two-metal ion mechanism. Unlike cellular RNases H, the retroviral RNases H display three different modes of cleavage: internal, DNA 3' end-directed, and RNA 5' end-directed. All three modes of cleavage appear to have roles in reverse transcription. Nucleotide sequence is an important determinant of cleavage specificity with both enzymes exhibiting a preference for specific nucleotides at discrete positions flanking an internal cleavage site as well as during tRNA primer removal and plus-strand primer generation. RNA 5' end-directed and DNA 3' end-directed cleavages show similar sequence preferences at the positions closest to a cleavage site. A model for how RNase H selects cleavage sites is presented that incorporates both sequence preferences and the concept of a defined window for allowable cleavage from a recessed end. Finally, the RNase H activity of HIV-1 is considered as a target for anti-virals as well as a participant in drug resistance.
Subject(s)
HIV-1/chemistry , HIV-1/enzymology , Moloney murine leukemia virus/chemistry , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/metabolism , Reverse Transcription , Ribonuclease H/chemistry , Ribonuclease H/metabolism , Amino Acid Sequence , Base Sequence , Crystallography, X-Ray , HIV Infections/drug therapy , HIV Infections/enzymology , HIV-1/drug effects , HIV-1/genetics , Humans , Moloney murine leukemia virus/drug effects , Moloney murine leukemia virus/genetics , Protein Structure, Secondary , Protein Structure, Tertiary , RNA-Directed DNA Polymerase/genetics , Reverse Transcriptase Inhibitors/pharmacology , Reverse Transcription/drug effects , Ribonuclease H/antagonists & inhibitors , Ribonuclease H/genetics , Substrate SpecificityABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) cleaves the phosphodiester bond between a covalently stalled topoisomerase I (Topo I) and the 3' end of DNA. Stalling of Topo I at DNA strand breaks is induced by endogenous DNA damage and the Topo I-specific anticancer drug camptothecin (CPT). The H493R mutation of Tdp1 causes the neurodegenerative disorder spinocerebellar ataxia with axonal neuropathy (SCAN1). Contrary to the hypothesis that SCAN1 arises from catalytically inactive Tdp1, Tdp1-/- mice are indistinguishable from wild-type mice, physically, histologically, behaviorally, and electrophysiologically. However, compared to wild-type mice, Tdp1-/- mice are hypersensitive to CPT and bleomycin but not to etoposide. Consistent with earlier in vitro studies, we show that the H493R Tdp1 mutant protein retains residual activity and becomes covalently trapped on the DNA after CPT treatment of SCAN1 cells. This result provides a direct demonstration that Tdp1 repairs Topo I covalent lesions in vivo and suggests that SCAN1 arises from the recessive neomorphic mutation H493R. This is a novel mechanism for disease since neomorphic mutations are generally dominant.
Subject(s)
Phosphoric Diester Hydrolases/metabolism , Spinocerebellar Ataxias/genetics , Animals , Antineoplastic Agents/pharmacology , Axons , Bleomycin/pharmacology , Brain/metabolism , Camptothecin/analogs & derivatives , Camptothecin/pharmacology , Cells, Cultured , Comet Assay , Embryo, Mammalian/cytology , Etoposide/pharmacology , Fibroblasts/drug effects , Fibroblasts/metabolism , Genes, Recessive , Humans , Irinotecan , Mice , Mice, Knockout , Mutation , Phosphoric Diester Hydrolases/deficiency , Phosphoric Diester Hydrolases/genetics , Polyneuropathies/genetics , Polyneuropathies/metabolism , RNA, Messenger/metabolism , Spinocerebellar Ataxias/metabolism , Topotecan/pharmacologyABSTRACT
A distinctive property of reverse transcriptase is the ability to carry out strand displacement synthesis in the absence of accessory proteins such as helicases or single-strand DNA binding proteins. Structure-function studies indicate that the fingers subdomain in HIV-1 reverse transcriptase contacts the template strand downstream of the primer terminus and is involved in strand displacement synthesis. Based on structural comparisons to the HIV-1 enzyme, we made single amino acid substitutions at the Tyr-64 and Leu-99 positions in the fingers subdomain of the M-MuLV reverse transcriptase to ask whether this subdomain has a similar role in displacement synthesis. In vitro assays comparing non-displacement versus displacement synthesis revealed that substitution of alanine at Tyr-64 generated a reverse transcriptase that was impaired in its capacity to carry out DNA and RNA displacement synthesis without affecting polymerase processivity or RNase H activity. However, substitution of Tyr-64 with phenylalanine and a variety of substitutions at position Leu-99 had no specific effect on displacement synthesis. The Y64A substitution prevented viral replication in vivo, and Y64A virus generated reduced levels of reverse transcription intermediates at all steps beyond the synthesis of minus strong stop DNA. The role of the fingers subdomain and in particular the possible contributions of the Tyr-64 residue in displacement synthesis are discussed.
Subject(s)
Amino Acid Substitution/genetics , DNA, Viral/metabolism , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/metabolism , Viral Proteins/metabolism , Virus Replication/genetics , Animals , Mice , Moloney murine leukemia virus/genetics , NIH 3T3 Cells , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , RNA, Viral/metabolism , RNA-Directed DNA Polymerase/genetics , Viral Proteins/geneticsABSTRACT
Type IB topoisomerases are essential enzymes that are responsible for relaxing superhelical tension in DNA by forming a transient covalent nick in one strand of the DNA duplex. Topoisomerase I is a target for anti-cancer drugs such as camptothecin, and these drugs also target the topoisomerases I in pathogenic trypanosomes including Leishmania species and Trypanosoma brucei. Most eukaryotic enzymes, including human topoisomerase I, are monomeric. However, for Leishmania donovani, the DNA-binding activity and the majority of residues involved in catalysis are located in a large subunit, designated TOP1L, whereas the catalytic tyrosine residue responsible for covalent attachment to DNA is located in a smaller subunit, called TOP1S. Here, we present the 2.27A crystal structure of an active truncated L.donovani TOP1L/TOP1S heterodimer bound to nicked double-stranded DNA captured as a vanadate complex. The vanadate forms covalent linkages between the catalytic tyrosine residue of the small subunit and the nicked ends of the scissile DNA strand, mimicking the previously unseen transition state of the topoisomerase I catalytic cycle. This structure fills a critical gap in the existing ensemble of topoisomerase I structures and provides crucial insights into the catalytic mechanism.
Subject(s)
DNA Topoisomerases, Type I/chemistry , DNA/chemistry , Leishmania donovani/enzymology , Protein Structure, Quaternary , Protozoan Proteins/chemistry , Vanadates/chemistry , Animals , Arginine/chemistry , Catalytic Domain , Crystallography, X-Ray , DNA/metabolism , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Dimerization , Histidine/chemistry , Humans , Macromolecular Substances , Models, Molecular , Molecular Structure , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Topoisomerase I Inhibitors , Tyrosine/chemistry , Water/chemistryABSTRACT
Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3' terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo.
Subject(s)
Recombination, Genetic , Retroviridae/genetics , Reverse Transcription , DNA, Viral/biosynthesis , DNA, Viral/genetics , Genes, Reporter , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Leukemia Virus, Murine/genetics , Nucleic Acid ConformationABSTRACT
The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5'-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5'-end. Previously, the distance from the RNA 5'-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5'-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5'-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the +1 position and C or G at the -2 position, and additionally for HIV-1, A is disfavored at the -4 position. For both enzymes, 5'-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5'-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5'-end, it was found that the extent of 5'-end-directed cleavages observed in substrates containing a free recessed RNA 5'-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5'-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5'-end.
Subject(s)
Retroviridae/enzymology , Ribonuclease H/chemistry , Base Sequence , Binding Sites , DNA/chemistry , DNA Primers/chemistry , HIV Reverse Transcriptase/genetics , Molecular Sequence Data , Moloney murine leukemia virus/enzymology , Oligonucleotides/chemistry , Protein Binding , RNA/chemistry , RNA/genetics , RNA-Directed DNA Polymerase/genetics , Retroviridae/genetics , Ribonuclease H/metabolism , Sequence Homology, Nucleic AcidABSTRACT
Human tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes the phosphodiester bond between a DNA 3' end and a tyrosyl moiety. In eukaryotic cells, this type of linkage is found in stalled topoisomerase I-DNA covalent complexes, and Tdp1 has been implicated in the repair of such complexes in vivo. We confirm here that the Tdp1 catalytic cycle involves a covalent reaction intermediate in which a histidine residue is connected to a DNA 3'-phosphate through a phosphoamide linkage. Most surprisingly, this linkage can be hydrolyzed by Tdp1, and unlike a topoisomerase I-DNA complex, which requires modification to be an efficient substrate for Tdp1, the native form of Tdp1 can be removed from the DNA. The spinocerebellar ataxia with axonal neuropathy neurodegenerative disease is caused by the H493R mutant form of Tdp1, which shows reduced enzymatic activity and accumulates the Tdp1-DNA covalent intermediate. The ability of wild type Tdp1 to remove the stalled mutant protein from the DNA likely explains the recessive nature of spinocerebellar ataxia with axonal neuropathy. In addition to its activity on phosphotyrosine and phosphohistidine substrates, Tdp1 also possesses a limited DNA and RNA 3'-exonuclease activity in which a single nucleoside is removed from the 3'-hydroxyl end of the substrate. Furthermore, Tdp1 also removes a 3' abasic site and an artificial 3'-biotin adduct from the DNA. In combination with earlier data showing that Tdp1 can use 3'-phosphoglycolate as a substrate, these data suggest that Tdp1 may function to remove a variety of 3' adducts from DNA during DNA repair.
Subject(s)
Phosphoamino Acids/chemistry , Phosphoric Diester Hydrolases/physiology , Alleles , Axons/pathology , Biotin/chemistry , Biotinylation , Catalysis , DNA/chemistry , DNA Repair , Furans/chemistry , Glycolates/chemistry , Humans , Hydrolysis , Models, Chemical , Models, Genetic , Mutation , Peptides/chemistry , Phosphoric Diester Hydrolases/metabolism , Protein Binding , RNA/chemistry , Substrate Specificity , Time Factors , Tyrosine/chemistryABSTRACT
Tyrosyl-DNA phosphodiesterase (Tdp1) catalyzes the hydrolysis of the tyrosyl-3' phosphate linkage found in topoisomerase I-DNA covalent complexes. The inherited disorder, spinocerebellar ataxia with axonal neuropathy (SCAN1), is caused by a H493R mutation in Tdp1. Contrary to earlier proposals that this disease results from a loss-of-function mutation, we show here that this mutation reduces enzyme activity approximately 25-fold and importantly causes the accumulation of the Tdp1-DNA covalent reaction intermediate. Thus, the attempted repair of topoisomerase I-DNA complexes by Tdp1 unexpectedly generates a new protein-DNA complex with an apparent half-life of approximately 13 min that, in addition to the unrepaired topoisomerase I-DNA complex, may interfere with transcription and replication in human cells and contribute to the SCAN1 phenotype. The analysis of Tdp1 mutant cell lines derived from SCAN1 patients reveals that they are hypersensitive to the topoisomerase I-specific anticancer drug camptothecin (CPT), implicating Tdp1 in the repair of CPT-induced topoisomerase I damage in human cells. This finding suggests that inhibitors of Tdp1 could act synergistically with CPT in anticancer therapy.
Subject(s)
Camptothecin/pharmacology , Enzyme Inhibitors/pharmacology , Phosphoric Diester Hydrolases/genetics , Amino Acid Substitution , Flow Cytometry , Humans , Mutation , Neoplasms/drug therapy , Phosphoric Diester Hydrolases/metabolism , S Phase/drug effectsABSTRACT
Human DNA topoisomerase I is an essential enzyme involved in resolving the torsional stress associated with DNA replication, transcription, and chromatin condensation. The catalytic cycle of the enzyme consists of DNA cleavage to form a covalent enzyme-DNA intermediate, DNA relaxation, and finally, re-ligation of the phosphate backbone to restore the continuity of the DNA. Structure/function studies have elucidated a flexible enzyme that relaxes DNA through coordinated, controlled movements of distinct enzyme domains. The cellular roles of topoisomerase I are apparent throughout the nucleus, but the concentration of processes acting on ribosomal DNA results in topoisomerase I accumulation in the nucleolus. Although the activity of topoisomerase I is required in these processes, the enzyme can also have a deleterious effect on cells. In the event that the final re-ligation step of the reaction cycle is prevented, the covalent topoisomerase I-DNA intermediate becomes a toxic DNA lesion that must be repaired. The complexities of the relaxation reaction, the cellular roles, and the pathways that must exist to repair topoisomerase I-mediated DNA damage highlight the importance of continued study of this essential enzyme.
Subject(s)
DNA Damage , DNA Topoisomerases, Type I/metabolism , DNA Replication , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/genetics , Humans , Models, Biological , Models, Molecular , Protein ConformationABSTRACT
Reverse transcriptase-mediated RNA displacement synthesis is required for DNA polymerization through the base-paired stem portions of secondary structures present in retroviral genomes. These regions of RNA duplex often possess single unpaired nucleotides, or "bulges," that disrupt contiguous base pairing. By using well defined secondary structures from the human immunodeficiency virus, type 1 (HIV-1), genome, we demonstrate that removal of these bulges either by deletion or by introducing a complementary base on the opposing strand results in increased pausing at specific positions within the RNA duplex. We also show that the HIV-1 nucleocapsid protein can increase synthesis through the pause sites but not as efficiently as when a bulge residue is present. Finally, we demonstrate that removing a bulge increases the proportion of strand transfer events to an acceptor template that occur prior to complete replication of a donor template secondary structure. Together our data suggest a role for bulge nucleotides in enhancing synthesis through stable secondary structures and reducing strand transfer.
Subject(s)
DNA, Viral/chemistry , HIV-1/metabolism , Nucleic Acid Conformation , Base Sequence , DNA/genetics , DNA Primers/chemistry , DNA Primers/genetics , Genome, Viral , HIV Reverse Transcriptase/genetics , Molecular Sequence Data , Plasmids/metabolism , RNA/chemistry , RNA/genetics , RNA/metabolism , Retroviridae/genetics , Templates, Genetic , Thermodynamics , Time Factors , Virus ReplicationABSTRACT
The RNase H activity of reverse transcriptase is essential to complete retroviral replication. Many studies have characterized how reverse transcriptase associates with recessed and exposed DNA 3' ends or RNA 5' ends to position the RNase H domain for cleavage, but little is known about how a nick might affect RNase H cleavages, or how RNase H carries out internal cleavages, which do not require positioning by a nucleic acid end. We have addressed these issues using model hybrid substrates and the reverse transcriptases of Moloney murine leukemia virus (M-MuLV) and human immunodeficiency virus type 1 (HIV-1). Our results show that a nick separating an upstream RNA and a downstream RNA annealed to DNA is essentially ignored by RNase H, indicating that the RNA 5' end at a nick is not sufficient to position 5' end-directed cleavages. Cleavage sites that are located close to the 5' end of the downstream RNA are not recognized in the absence of the upstream RNA, and the 5' ends of the shorter upstream RNAs enhance cleavage at these sites. The recognition of an internal cleavage site depends on local sequence features found both upstream and downstream of the cleavage site, designated as the -1/+1 position. By analyzing the nucleotide frequencies in the sequence surrounding strong internal cleavage sites, preferred nucleotides have been identified in the flanking sequences spanning positions -14 to +1 for HIV-1 and -11 to +1 for M-MuLV. These data reveal that general degradation of the retroviral genome after minus-strand synthesis can occur through sequence-specific cleavages.
Subject(s)
HIV-1/enzymology , Moloney murine leukemia virus/enzymology , Ribonuclease H/metabolism , Base Sequence , DNA Primers , HIV-1/physiology , Hydrolysis , Moloney murine leukemia virus/physiology , Phosphorylation , Substrate Specificity , Virus ReplicationABSTRACT
DNA displacement synthesis by reverse transcriptase during retroviral replication is required for the production of the linear precursor to integration. The sensitivity of unpaired thymines to KMnO(4) oxidation was used to probe for the extent of DNA melting by human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase in front of the primer terminus in model oligonucleotide-based displacement constructs. Unpairing of the two base pairs downstream of the primer (+1 and +2 positions) requires the presence of the next correct dNTP, indicating that DNA melting only occurs after the formation of the ternary complex with the enzyme tightly clamped around the DNA. The amount or extent of DNA melting is not significantly affected by the length of the already-displaced strand or the base composition of the DNA beyond the +2 position. The F61W mutant form of HIV-1 reverse transcriptase, which is partially impaired for displacement synthesis, exhibits a reduction in the amount of melting at the +1 and +2 positions. These results demonstrate the importance of the observed melting to displacement synthesis and suggest that the unpairing reaction is mediated by an intimate association between the fingers region of the enzyme and the DNA in the closed clamp conformation of the protein.
Subject(s)
DNA, Viral , DNA/genetics , HIV Reverse Transcriptase/genetics , Potassium Permanganate/chemistry , Base Sequence , DNA/chemistry , DNA Primers/chemistry , Dimerization , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Models, Genetic , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Oligonucleotides/chemistry , Thymine/chemistry , Time FactorsABSTRACT
During reverse transcription of viral RNA, HIV-1 reverse transcriptase (RT) encounters RNA stem-loop structures that require displacement synthesis activity in which RT disrupts the RNA helix to access the template strand. A primer extension assay was developed to assess HIV-1 RT RNA displacement synthesis activity in vitro. Initial results revealed that HIV-1 RT performs only limited amounts of RNA displacement through long stretches of RNA duplex, with the majority of synthesis stalling at sequence-dependent pause positions. DNA displacement synthesis through the same sequence, however, proceeded rapidly to the end of the template. The RNA folding algorithm mfold indicated that the presence of an unpaired nucleotide, or "bulge," along the RNA duplex would promote helix melting ahead of the DNA primer terminus to create a small gap of nondisplacement synthesis. Primer extension assays using substrates possessing single-nucleotide bulges in the nontemplate strand near pause sites resulted in diminished pausing at positions within the predicted melted region. Surprisingly, the bulges also reduced pausing distal to the bulge at positions that are expected to remain base-paired. Further analysis revealed that stalling during RNA displacement synthesis results from the displaced RNA re-annealing to the template strand thus forcing the primer terminus to become unpaired and, therefore, not extendable. Introduction of a bulge facilitates displacement synthesis through distal regions by increasing RT processivity in the vicinity of a bulge and reducing the impact of branch migration on primer extension.