Biomedical subjects

J J Champoux

Publications and source records attributed to J J Champoux.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of RNA strand displacement synthesis by Moloney murine leukemia virus reverse transcriptase.

The RNase H activity of reverse transcriptase (RT) is presumably required to cleave the RNA genome following minus strand synthesis to free the DNA for use as a template during plus strand synthesis. However, since RNA degradation by RNase H appears to generate RNA fragments too large to spontaneously dissociate from the minus strand, we have investigated the possibility that RNA displacement by RT during plus strand synthesis contributes to the removal of RNA fragments. By using an RNase H- mutant of Moloney murine leukemia virus (M-MuLV) RT, we demonstrate that the polymerase can displace long regions of RNA in hybrid duplex with DNA but that this activity is approximately 5-fold slower than DNA displacement and 20-fold slower than non-displacement synthesis. Furthermore, we find that although certain hybrid sequences seem nearly refractory to the initiation of RNA displacement, the same sequences may not significantly impede synthesis when preceded by a single-stranded gap. We find that the rate of RNA displacement synthesis by wild-type M-MuLV RT is significantly greater than that of the RNase H- RT but remains less than the rate of non-displacement synthesis. M-MuLV nucleocapsid protein increases the rates of RNA and DNA displacement synthesis approximately 2-fold, and this activity appears to require the zinc finger domain.

Base Sequence↗

PubMed1998

Crystal structures of human topoisomerase I in covalent and noncovalent complexes with DNA.

Topoisomerases I promote the relaxation of DNA superhelical tension by introducing a transient single-stranded break in duplex DNA and are vital for the processes of replication, transcription, and recombination. The crystal structures at 2.1 and 2.5 angstrom resolution of reconstituted human topoisomerase I comprising the core and carboxyl-terminal domains in covalent and noncovalent complexes with 22-base pair DNA duplexes reveal an enzyme that "clamps" around essentially B-form DNA. The core domain and the first eight residues of the carboxyl-terminal domain of the enzyme, including the active-site nucleophile tyrosine-723, share significant structural similarity with the bacteriophage family of DNA integrases. A binding mode for the anticancer drug camptothecin is proposed on the basis of chemical and biochemical information combined with these three-dimensional structures of topoisomerase I-DNA complexes.

Antineoplastic Agents, Phytogenic↗

PubMed1998

A model for the mechanism of human topoisomerase I.

The three-dimensional structure of a 70-kilodalton amino terminally truncated form of human topoisomerase I in complex with a 22-base pair duplex oligonucleotide, determined to a resolution of 2.8 angstroms, reveals all of the structural elements of the enzyme that contact DNA. The linker region that connects the central core of the enzyme to the carboxyl-terminal domain assumes a coiled-coil configuration and protrudes away from the remainder of the enzyme. The positively charged DNA-proximal surface of the linker makes only a few contacts with the DNA downstream of the cleavage site. In combination with the crystal structures of the reconstituted human topoisomerase I before and after DNA cleavage, this information suggests which amino acid residues are involved in catalyzing phosphodiester bond breakage and religation. The structures also lead to the proposal that the topoisomerization step occurs by a mechanism termed "controlled rotation."

Amino Acid Sequence↗

PubMed1998

Domains of human topoisomerase I and associated functions.

Human topoisomerase I can be divided into four domains based on homology alignments, physical properties, sensitivity to limited proteolysis, and fragment complementation studies. Roughly the first 197 amino acids represent the N-terminal domain that appears to be devoid of secondary structure and is likely important for targeting the enzyme to its sites of action within the nucleus of the cell. The core domain encompasses residues approximately 198 to approximately 651, is involved in catalysis, and is important for the preferential binding of the enzyme to supercoiled DNA. The C-terminal domain extends from residue approximately 697 to the end of the protein at residue 765 and contains the catalytically important active site tyrosine at position 723. The core and C-terminal domains are connected by a poorly conserved, protease-sensitive linker domain (residues approximately 652 to approximately 696) that has been implicated in DNA binding and may influence how long the enzyme remains in the nicked stated. Mutations that confer resistance to the topoisomerase I poison camptothecin are located in the core and C-terminal domains and presumably identify residues important for drug binding.

Binding Sites↗

PubMed1997

Reconstitution of human topoisomerase I by fragment complementation.

Human topoisomerase I (topo I, 91 kDa) is composed of four major domains; the unconserved and highly charged "N-terminal" domain (24 kDa), the conserved "core" domain (54 kDa), a poorly conserved and positively charged "linker" region (5 kDa), and the highly conserved "C-terminal" domain (8 kDa) which contains the active site tyrosine at position 723. Here we demonstrate that human topo I activity can be reconstituted by mixing a 58 kDa recombinant core domain (residues Lys175 to Ala659) with any one of a series of recombinant C-terminal fragments that range in size from 12 kDa (linker and C-terminal domains, residues Leu658 to Phe765) to 6.3 kDa (C-terminal domain residues Gln713 to Phe765). The C-terminal fragments bind tightly to the core domain, forming a 1:1 complex that is stable irrespective of ionic strength (0.01 to 1 M). The reconstituted enzymes are active only over a relatively narrow range of salt concentrations (25 to 200 mM KCl) as compared to the intact topo70 enzyme (missing the N-terminal domain). Under physiological conditions (150 mM KCl and 10 mM Mg2+) they are much more distributive in their mode of action than topo70. The reconstituted enzyme binds DNA with an affinity that is approximately 20-fold lower than that of the intact topo70. In addition, the cleavage/religation equilibrium of the reconstituted enzyme appears to be biased towards religation relative to that of the intact enzyme. Despite differences in the cleavage/religation equilibrium and affinity for DNA, the reconstituted and intact enzymes have identical sequence specificities for the cleavage of duplex DNA or suicide cleavage of oligonucleotide substrates.

Animals↗

PubMed1997

Topoisomerase I enhances TFIID-TFIIA complex assembly during activation of transcription.

The mechanism of coactivation by DNA topoisomerase I (topo I) was examined in a highly defined in vitro transcription system containing Pol II and purified factors. Both stimulation of the basal reaction and coactivation occurred dependent on TAF(II)s. Activation was first observed at the TFIID-TFIIA stage of initiation and maximal activation required the concomitant presence of TFIID, TFIIA, topo I, and activator. Electrophoretic mobility shift assay demonstrated a dramatic enhancement in the formation of the TFIID-TFIIA complex by topo I and activator, dependent on the TAF(II)s. DNase I footprinting confirmed this recruitment. A catalytically inactive topo I, which coactivated transcription, similarly stimulated the rapid formation of the TFIID-TFIIA complex in the presence of activator. A camptothecin-mediated DNA cleavage assay demonstrated the recruitment of topo I to the template by TFIID. Topo I likely functions during activation by enhancing the formation of an active TFIID-TFIIA complex on the promoter.

Camptothecin↗

PubMed1996

Biochemical and biophysical analyses of recombinant forms of human topoisomerase I.

Amino acid sequence comparisons of human topoisomerase I (Topo I) with seven other cellular Topo I enzymes reveal that the enzyme can be divided into four major domains: the unconserved NH2-terminal domain (24 kDa), the conserved core domain (54 kDa), a poorly conserved linker region (5 kDa), and the highly conserved COOH-terminal domain (8 kDa), which contains the active site tyrosine. To investigate this predicted domain organization, recombinant baculoviruses were engineered to express the 91-kDa full-length enzyme, a 70-kDa NH2-terminally truncated enzyme that is missing the first 174 residues, and a 58-kDa NH2- and COOH-terminally truncated core fragment encompassing residues 175-659. The specific activity of the full-length and Topo70 enzymes are indistinguishable from the native human Topo I purified from HeLa cells. Each protein is inhibited by camptothecin, topotecan, and 9-aminocamptothecin, but not by ATP. Activity is stimulated by Mg2+, Ba2+, Ca2+, Mn2+, spermine, and spermidine. The magnitude of the stimulatory effect of Mg2+ is inversely proportional to the salt concentration. Furthermore, at KCl concentrations of 300 mM or greater, the addition of Mg2+ is inhibitory. The effects of Mg2+ and the polycations spermine and spermidine are partially additive, an indication that the stimulatory mechanisms of the two substances are different. Activity was strongly inhibited or abolished by Ni2+, Zn2+, Cu2+, Cd2+, and Co2+. An examination of the hydrodynamic properties of full-length Topo I, Topo70, and Topo58 demonstrates that the core, linker, and COOH-terminal domains fold into a globular structure, while the NH2-terminal domain is highly extended. A comparison of the circular dichroism spectra of full-length Topo I and Topo70 demonstrates that residues 1-174 (approximately 21 kDa) of Topo I are largely if not completely unfolded. This observation is consistent with the fact that the NH2-terminal domain is dispensable for activity.

Adenosine Triphosphate↗

PubMed1996

The domain organization of human topoisomerase I.

Using limited proteolysis, we show that the domain boundaries of human topoisomerase I closely parallel those predicted from sequence comparisons with other cellular Topo I enzymes. The enzyme is comprised of (i) an NH2-terminal domain (approximately 24 kDa), which is known to be dispensable for activity, (ii) the core domain (approximately 54 kDa), (iii) a linker region (approximately 3 kDa), and (iv) the COOH-terminal domain (approximately 10 kDa), which contains the active site tyrosine. The highly conserved core and COOH-terminal domains are resistant to proteolysis, while the unconserved NH2-terminal and linker domains are sensitive. Noncovalent binding of Topo I to plasmid DNA or to short duplex oligonucleotides decreases the sensitivity of the linker to proteolysis by approximately a factor of 10 but has no effect on proteolysis of the NH2-terminal domain. When the enzyme is covalently complexed to an 18 base pair single-stranded oligonucleotide, the linker region is sensitive to proteolysis whether or not duplex DNA is present. The net positive charge of the linker domain suggests that at a certain point in catalysis the linker may bind directly to DNA. Further, we show that limited subtilisin cleavage can generate a mixture of 60-kDa core and approximately 10-kDa COOH-terminal fragments, which retain a level of topoisomerase activity that is nearly equal to undigested control samples, presumably because the two fragments remain associated after proteolytic cleavage. Thus, despite its potential role in DNA binding, the linker domain (in addition to the NH2-terminal domain) appears to be dispensable for topoisomerase activity. Finally, the limited proteolysis pattern of the human enzyme differs substantially from the limited proteolysis pattern of the vaccinia viral Topo I, indicating that the two enzymes belong to separate eukaryotic topoisomerase I subfamilies.

Base Sequence↗

PubMed1996

RNase H domain of Moloney murine leukemia virus reverse transcriptase retains activity but requires the polymerase domain for specificity.

The reverse transcriptase-associated RNase H activity of Moloney murine leukemia virus specifically cleaves within the polypurine tract region of the viral genome to generate the primer for plus-strand DNA synthesis and removes the tRNA primer after minus-strand initiation by preferentially cleaving the RNA one nucleotide before the RNA-DNA junction. Moreover, the enzyme is unable to cleave the extended tRNA substrate at the RNA-DNA junction even at high enzyme concentrations. The RNase H domain of the reverse transcriptase was expressed as a glutathione S-transferase fusion protein and purified from Escherichia coli extracts. Following removal of the glutathione S-transferase portion of the protein, the specificity of the isolated RNase H domain was determined in the plus-strand primer reaction and in the tRNA primer removal reaction. Although the isolated domain lacked specificity in both cases, it was still unable to cleave the tRNA substrate precisely at the RNA-DNA junction. Specificity in both cases could be restored by adding back a truncated form of Moloney murine leukemia virus reverse transcriptase lacking the RNase H domain. These results implicate the polymerase domain as a specificity determinant for the RNase H activity of reverse transcriptase. The isolated RNase H domain had higher activity in the presence of Mn2+ than in the presence of Mg2+, but neither the RNase H domain alone nor the RNase H domain coupled to the polymerase domain in wild-type protein exhibited the normal cleavage specificities in the presence of the nonphysiological divalent cation.

DNA-Directed DNA Polymerase↗

PubMed1995

Preferential binding of human topoisomerase I to superhelical DNA.

Eukaryotic type I DNA topoisomerase provides swivels for removing torsional strain from the DNA helix during transcription and DNA replication. Previously it has been shown that the enzyme is associated with actively transcribed genes and replicating DNA. Using an inactive mutant form of the protein containing phenylalanine instead of tyrosine at position 723, we have investigated the binding properties of the protein as a function of substrate topology. A series of filter binding assays indicated that the protein strongly prefers to bind superhelical over completely relaxed SV40 DNA. The ability of a supercoiled DNA to compete against a relaxed DNA for binding increases as the number of superhelical turns in the DNA increases. Since positively supercoiled DNA is bound with the same preference as negatively supercoiled DNA, we hypothesize that topoisomerase I binds preferentially at the nodes created by the crossing of two duplex helices. The preference for binding superhelical DNA is also exhibited by the conserved core domain (amino acids 175-659) which is missing the active site region located near the C-terminus. These results suggest that this core domain may target the enzyme in vivo to regions of torsionally strained superhelical DNA.

Binding Sites↗

PubMed1995

Cleavage specificities of Moloney murine leukemia virus RNase H implicated in the second strand transfer during reverse transcription.

Reverse transcription of a retroviral RNA genome requires two template jumps to generate the linear double-stranded DNA required for integration. The RNase H activity of reverse transcriptase has several roles during this process. We have examined RNase H cleavages that define the maximal 3' and 5' ends of Moloney murine leukemia virus minus strand DNA prior to the second template jump. In both the endogenous reaction and on model substrates in vitro, RNase H cleaves the genomic RNA template between the second and third ribonucleotides 5' of the U5/PBS junction, but other minor cleavages between 1 and 10 nucleotides 5' of this junction are also observed. Similar experiments examining the specificity of RNase H for tRNA primer removal revealed that cleavage generally leaves a ribo A residue at the 5' end of minus strand DNA. These observations suggest that three bases are typically duplicated on the ends of the minus strands, leading to an intermediate following the second jump which contains unpaired nucleotides. Model substrates mimicking the structure of this intermediate demonstrate that reverse transcriptase has little difficulty in utilizing such a branched structure for the initiation of displacement synthesis.

Animals↗

PubMed1995

Detection of anti-topoisomerase I antibodies using a full length human topoisomerase I recombinant protein purified from a baculovirus expression system.

Topoisomerase I (topo I) is a major systemic sclerosis (SSc)-associated autoantigen. A cDNA construct encoding full length human topo I in a recombinant baculovirus transfer vector was used to infect insect cells in culture from which recombinant protein was purified. An ELISA using recombinant protein was evaluated in 340 sera including sera from 134 patients with SSc, of whom 33 had anti-topo I antibodies detected by immunodiffusion. A high yield of pure topo I of expected molecular mass and catalytic activity was obtained. The recombinant topo I ELISA was 92% sensitive and 98% specific in detecting anti-topo I antibodies which were present almost exclusively in patients with SSc. Therefore, the potential advantages of expressing human autoantigens in eukaryotic systems for diagnostic purposes were confirmed.

Animals↗

PubMed1994

The use of DNA and RNA oligonucleotides in hybrid structures with longer polynucleotide chains to probe the structural requirements for moloney murine leukemia virus plus strand priming.

Plus strand priming during retroviral reverse transcription requires specific cleavage within the polypurine tract of the viral genome by the reverse transcriptase-associated RNase H. Previously it has been shown that a 190-base RNA-DNA hybrid containing the Moloney murine leukemia virus polypurine tract can serve as a substrate for the priming reaction. To investigate the structural requirements for the reaction, a series of DNA oligonucleotides was hybridized to the 190-base single-stranded RNA and tested as substrates for RNase H. At low enzyme concentrations, the sites of cleavage are located 17-23 nucleotides from the 3'-end of the DNA oligonucleotide, consistent with the observations of others that binding of the DNA polymerase at a primer terminus fixes the position of cleavage by RNase H. At higher enzyme concentrations, additional cleavages are observed in the RNA 3' of these sites, but there is no preference for cleavage at the plus strand origin. In contrast to the results with DNA oligonucleotides, hybridization of RNA oligonucleotides containing the polypurine tract to the 190-base single-stranded DNA generates substrates that are cleaved at the origin and efficiently extended into DNA. An RNA oligonucleotide hybridized downstream of the polypurine tract is cleaved but not extended. These results support the view that RNase H cleavage to generate the plus strand primer is uncoupled from minus strand DNA synthesis.

Base Sequence↗

PubMed1994

Mechanism of catalysis by eukaryotic DNA topoisomerase I.

The elucidation of the chemistry of the topo I reaction has provided the first example of how a phosphodiester bond in DNA can be temporarily broken and the energy for reclosure stored in a covalent linkage between the end of the broken strand and the enzyme (Champoux, 1977a, 1981). This type of reaction offers several advantages to the cell. First, unnecessary exposure of DNA ends to nucleolytic attack is prevented. Second, breakage and reclosure of DNA strands can occur without an expenditure of ATP energy. Third, the combined breakage and rejoining reactions can be both spatially and temporally coordinated with other cellular activities by regulating the activity of a single protein molecule. This general mechanism has not only been extended to type II topoisomerases (see Chapters 3 and 5), but also to the specialized single-stranded phage replication proteins (e.g., phi X174 gene A protein) (Ikeda et al., 1976; Eisenberg et al., 1977) and to site-specific recombinases such as the bacteriophage lambda integrase (Craig and Nash, 1983), the delta gamma and Tn3 resolvases (Reed, 1981; Reed and Grindley, 1981; Krasnow and Cozzarelli, 1983; Hatfull and Grindley, 1986), and the yeast 2-microns circle FLP recombinase (Andrews et al., 1985; Gronostajski and Sadowski, 1985). Since the site-specific recombinases attach the broken strand to a different terminus rather than simply restoring the original phosphodiester bond as conventional topoisomerases do, they have been referred to as DNA strand transferases. It is conceivable that a similar mechanism applies to the rearrangement of immunoglobulin genes (Schatz et al., 1990) and to other specific genomic rearrangements that might occur during development (Matsuoka et al., 1991).

Animals↗

PubMed1994

Strand displacement synthesis capability of Moloney murine leukemia virus reverse transcriptase.

The accepted model of retroviral reverse transcription includes a circular DNA intermediate which requires strand displacement synthesis for linearization and creation of an integration-competent, long terminal repeat-flanked DNA product. We have used an in vitro model of this last step of reverse transcription to examine the role of the viral enzyme, reverse transcriptase (RT), in displacement synthesis. We show that Moloney murine leukemia virus RT possesses an activity which allows for displacement synthesis through a minimum of 1,334 bp of duplex DNA--an extent much greater than that required during in vivo reverse transcription and over 25-fold greater than has been previously demonstrated for a viral RT. RT does not function as a helicase in the classical sense but appears to closely couple duplex DNA melting with synthesis-driven translocation of the enzyme. In the absence of synthesis, the unwound region created by a primer-positioned RT appears to be no greater than 2 bp and does not advance along the template. Additionally, RT does not utilize ATP or any deoxynucleoside triphosphate not directly encoded by the template strand to catalyze processive duplex unwinding at a nick; nor does binding of the enzyme unwind duplex DNA in the absence of a 3' terminus. The approximate maximum chain elongation rate during strand displacement synthesis by Moloney murine leukemia virus RT falls between 0.73 and 1.5 nucleotides per s at 37 degrees C. The RNase H activity of RT does not appear to play a role in displacement synthesis; however, a 181-amino-acid C-terminal truncation of RT displays a dramatically reduced ability to catalyze synthesis through duplex DNA.

Base Sequence↗

PubMed1993

DNA topoisomerase I is involved in both repression and activation of transcription.

Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein.

Amino Acid Sequence↗

PubMed1993

Analysis of the biased distribution of topoisomerase I break sites on replicating simian virus 40 DNA.

We previously mapped the locations of breaks introduced by eukaryotic topoisomerase I (topo I) in replicating simian virus 40 (SV40) DNA and observed an approximate 3:1 bias in the distribution of the break sites for the template strand for discontinuous DNA synthesis. In the present study, this bias has been confirmed by the mapping of additional sites utilizing a standard primer extension assay and a sensitive repetitive primer extension (RPE) method. No new sites could be detected on either strand of SV40 by the RPE method, despite the 10 to 20 fold greater sensitivity of the technique. To investigate the nature of the bias, a detailed analysis of the SV40 DNA sequence was undertaken. A set of 17 pentanucleotide sequences derived from those sites observed to be broken in the viral DNA extracted from SV40-infected cells define an in vivo consensus sequence. We show that the observed strand bias is likely due to the intrinsic asymmetric distribution of these consensus sequences on the two strands of SV40 DNA. To confirm these observations, double-stranded oligonucleotides containing previously identified in vivo topo I break sites were introduced in both orientations into SV40 to generate insertion mutants. Mapping experiments utilizing these mutants revealed that the inserted topo I break sites were broken in vivo regardless of their orientation, confirming that the SV40 sequence is the major, if not the sole determinant, of the observed strand bias. The possible origins of the strand bias are discussed in relation to the evolution of the virus.

Animals↗

PubMed1993

The sequence features important for plus strand priming by human immunodeficiency virus type 1 reverse transcriptase.

A specific cleavage by the reverse transcriptase-associated RNase H activity generates the RNA primer for plus strand DNA synthesis during reverse transcription. Previously, we used site-directed mutagenesis to define the sequence features of the polypurine tract (PPT) required for correct plus strand priming by the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Rattray, A. J., and Champoux, J. J. (1989) J. Mol. Biol. 208, 445-456). Although the sequences of human immunodeficiency virus type 1 (HIV-1) and M-MuLV diverge completely outside a 20-base region encompassing the PPT, within this region there are only three differences between the two viruses. Here we show that the HIV-1 reverse transcriptase will utilize the M-MuLV PPT as an origin for plus strand initiation in vitro. This finding enabled us to use the set of PPT mutants previously generated in M-MuLV, in conjunction with a small set of newly derived mutations within the HIV-1 PPT, to study plus strand priming by the HIV-1 reverse transcriptase. Despite the similarity between the two PPT regions, the sequence features important for positioning RNase H for the cleavage reaction that generates the plus strand primer are different for the two viruses. For M-MuLV, the -7A residue is a critical specificity determinant in the priming reaction, whereas for HIV-1, the -2G and -4G residues play key roles in determining the specificity of priming.

Base Sequence↗