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J J Champoux

Publications and source records attributed to J J Champoux.

At least 73 records · Page 4Linked to original sources
PubMed1984

Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA.

The site specificities of the type 1 DNA topoisomerases (topo 1) from rat liver and wheat germ were investigated. The nucleotide sequence at break sites on duplex SV40 DNA were determined for 245 wheat germ topo 1 sites and 223 rat liver topo 1 sites over a region of 1781 nucleotides. The enzymes from the two different sources show similar, but not identical patterns of DNA strand breakage. The sites occur frequently, but are not broken with equal probabilities. Major sites of breakage occur on the average every one to two turns of the helix, thus if sites of breakage accurately represent topo 1 sites of activity, the DNA sequence alone would appear to place few limits on the access of the enzyme to DNA. Sequences around the strongest sites for both enzymes show a bias in base composition for the four nucleotides immediately 5' to the break site (-4 to -1 positions), but no bias is observed 3' to the site of breakage. Consensus sequences for both enzymes were determined. Variations from the consensus sequence appear to affect the two enzymes differently and may account for the differences observed in the specificity of breakage.

Animals↗
PubMed1984

Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase.

The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.

Animals↗
PubMed1984

RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro.

A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well.

Base Sequence↗
PubMed1984

Efficient misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate.

Misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate was studied using 32P-labeled DNA primers annealed to the appropriate template DNA, and polyacrylamide-urea gel electrophoresis to measure the extension of the primer chains. With most primer-template combinations, greater than 50% of the primers were extended by the addition of a single incorrect nucleotide onto the end of the primer chain. Unexpectedly, one primer-template combination was not extended in the presence of dCTP, although misincorporation occurred with the other deoxyribonucleoside triphosphates. In another case, terminal misincorporation of two rather than one dT residue was observed. The primer termini containing unpaired nucleotides were efficiently extended upon addition of the other three deoxyribonucleoside triphosphates, even in the case where the primer terminus contained two unpaired nucleotides. Misincorporation was confirmed by direct sequence analysis. These results indicate that the frequency of mutations following misincorporation by reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate should be sufficiently high to allow detection of mutants by simple screening procedures. An analysis of the sequence of a mutant resulting from misincorporation at the M13mp2 AvaII site revealed that following introduction of the DNA into Escherichia coli cells, mismatch repair preceded replication.

Avian Leukosis Virus↗
PubMed1983

A cellular single-stranded DNA-dependent ATPase associated with simian virus 40 chromatin.

A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.

Adenosine Triphosphatases↗
PubMed1981

The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin.

The optimum monovalent cation concentration (Na+ or K+) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150-200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalent cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking -closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.

Animals↗
PubMed1981

DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine.

Conditions which result in DNA strand breakage by the rat liver DNA nicking-closing enzyme lead to the covalent attachment of the 3'-end of the broken strand to the enzyme. Treatment of this complex with pancreatic DNase leaves a residue of 17 +/- 8 nucleotide phosphates still attached to the enzyme. Subsequent nuclease P1 treatment removes all but 2 +/- 1 phosphate residues. Using nuclease P1-treated complexes which had been labeled in the DNA with 32P, the stability of the protein-DNA linkage was studied. The linkage is stable to acid, base, neutral and acidic hydroxylamine, and neutral I2. This pattern of stability rules out essentially all of the possible DNA-protein linkages except for a linkage involving a phosphodiester bond to the amino acid tyrosine. After acid hydrolysis of the 32P-labeled complexes, label was found to be associated with O4-phosphotyrosine, providing a direct demonstration that tyrosine is the amino acid to which the end of the DNA chain is attached.

Animals↗
PubMed1981

DNA breakage and closure by rat liver type 1 topoisomerase: separation of the half-reactions by using a single-stranded DNA substrate.

Circular single strands of bacteriophage phi X174 DNA are broken by rat liver DNA nicking-closing enzyme (type 1 topoisomerase) in low salt (50 mM KCl) at 37 degrees C, generating linear strands containing covalently bound enzyme [Been, M. D. & Champoux, J. J. (1980) Nucleic Acids Res. 8, 6129-6142]. The linear strands can be recircularized in the presence of 10 mM MgCl2 at 24 degrees C and 37 degrees C or 250 mM KCl at 24 degrees C. Recircularization is blocked when the hydroxyl group at the 5' terminus is phosphorylated. The linears generated by the nicking-closing enzyme can also be joined to other DNA fragments containing 5' hydroxyls, but not 5' phosphates. The linkage formed in both the intrastrand and interstrand reactions is stable to alkali. Reclosure of broken single strands is presumed to be analogous to the closure step that occurs durng nicking and closing cycles on duplex DNA.

Animals↗
PubMed1980

Breakage of single-stranded DNA by rat liver nicking-closing enzyme with the formation of a DNA-enzyme complex.

The DNA nicking-closing enzyme (type I topoisomerase) from rat liver nuclei breaks single-stranded DNA. The broken strand contains a 5'-hydroxyl and tightly bound protein. The stability of this protein-DNA complex to high salt, alkali and detergent suggests a covalent linkage between the DNA and the enzyme. The observed breakage of single-stranded DNA occurs at neutral pH prior to treatment with alkali or detergent, indicating that the breakage may be the result of an interrupted nicking and closing cycle. The resulting covalent complex could represent a reaction intermediate in the overall nicking-closing reaction.

Animals↗
PubMed1978

The DNA untwisting enzyme from Saccharomyces cerevisiae. Partial purification and characterization.

The DNA untwisting enzyme has been partially purified from Saccharomyces cerevisiae. The enzyme exhibits a pH optimum of 7.3 to 7.6 in phosphate buffer, appears to require 0.15 M KCl for activity as determined by a DNA filter-binding assay, and is inhibited by N-ethylmaleimide. Like the untwisting enzymes from other eucaryotic cells, it can remove both positive and negative superhelical turns. A DNA molecule containing a single strand break was shown to be an intermediate in the untwisting reaction. Thermal stabilities of the enzyme from selected conditional lethal mutants defective in DNA synthesis have been examined and were found to be indistinguishable from the wild type enzyme.

DNA Topoisomerases, Type I↗
PubMed1978

Interaction of the DNA untwisting enzyme with the SV40 nucleoprotein complex.

The SV40 nucleoprotein complex which was isolated from infected CV-1 cells did not possess an active DNA untwisting enzyme. The superhelix density of the DNA in the chromatin complex was unchanged after treatment with purified rat liver DNA untwisting enzyme. However, in the presence of ethidium bromide (1 microgram/ml) the superhelix density was changed. Moreover, the nicked intermediate in the DNA untwisting reaction could be detected using the chromatin DNA as a substrate. These results show that the DNA in the SV40 chromatin which is accessible to the DNA untwisting enzyme is under no topological strain.

Chromatin↗
PubMed1978

Proteins that affect DNA conformation.

In eucaryotic cells virtually all of the DNA is complex with proteins to form a unit fiber approximately 100 A in diameter. Chromatin is formed by the higher order coiling of the unit fiber. In procaryotic cells, as exemplified by E. coli, the actual structure of the chromosome is less clear (218), but the discovery of the DNA gyrase raises the possibility that the DNA helix in the cell is maintained in an underwound state. It may be important to consider these structural features of DNA in future biochemical studies on replication, transcription, and recombination. The recent discoveries of the DNA swivelases, the DNA gyrase, and the DNA unwinding enzymes considerably increase our knowledge of DNA biochemistry. As more is learned about these enzymes and their interaction with DNA, the prospects for understanding the details of DNA transcription, DNA recombination, and particularly DNA replication appear to be good.

Bacterial Proteins↗
PubMed1977

Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme.

Renaturation of two complementary single-stranded circles should be limited by topological constraints against the rewinding of the DNA helix. If a mixture of complementary single-stranded rings is annealed and then treated with the DNA untwisting enzyme, the DNA circles completely renature as judged by (i) the presence of interlocked rings that sediment at 53 S in alkali, (ii) the buoyant density of the renatured DNA in CsCl gradients containing ethidium bromide, and (iii) the resistance of the product to the single-strand-specific S1 nuclease. Therefore, the DNA untwisting enzyme is able to provide a transient single-strand break that is sufficient to allow the two strands to completely rewind. The possibility that the untwisting enzyme might facilitate the initiation of the process of genetic recombination is discussed.

DNA Topoisomerases, Type I↗