A molecular switch in a replication machine defined by an internal competition for protein rings.

Replication machines use ring-shaped clamps that encircle DNA to tether the polymerase to the chromosome. The clamp is assembled on DNA by a clamp loader. This report shows that the polymerase and clamp loader coordinate their actions with the clamp by competing for it through overlapping binding sites. The competition is modulated by DNA. In the absence of DNA, the clamp associates with the clamp loader. But after the clamp is placed on DNA, the polymerase develops a tight grip on the clamp and out-competes the clamp loader. After replication of the template, the polymerase looses affinity for the clamp. Now the clamp loader regains access to the clamp and removes it from DNA thus recycling it for future use.

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. I. Organization of the clamp loader.

The gamma complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of beta sliding clamps onto primed DNA. The beta sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis. In this report, the gamma complex has been constituted from its five different subunits. Size measurements and subunit stoichiometry studies show a composition of gamma 2 delta 1 delta' 1 1 chi 1 psi 1. Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements. An analogous tau complex has also been constituted and characterized; it is nearly as active as the gamma complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the delta, delta', chi, and psi subunits when core is present (Xiao, H., Naktinis, V., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13378-13383). The single copy subunits within the gamma complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme.

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp.

The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles DNA and tethers the replicase to the template. The beta dimer is assembled around DNA by the gamma complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and beta is identified as mediated through the delta subunit of the gamma complex. The delta subunit appears to contact the face of the beta dimer ring that contains the two C termini. Surprisingly, ATP is required for the gamma complex to bind beta, but not for delta to bind beta. This indicates that delta is buried in the gamma complex and suggests a role for ATP in exposing delta for interaction with beta. A protease protection assay has been developed to specifically probe the delta subunit within the gamma complex. The results of the assay are consistent with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it. The implication of these key features to the clamp loading mechanism of the gamma complex is discussed.

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. III. Interface between two polymerases and the clamp loader.

The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha 2 epsilon 2 theta 2 tau 2 gamma 2 delta 1 delta' 1 chi 1 psi 1. The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a dimer of tau and one gamma complex clamp loader (gamma 2 delta 1 delta' 1 chi 1 psi 1) for loading beta onto DNA. At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the delta, delta', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. IV. ATP-binding site mutants identify the clamp loader.

The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA. The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits. Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau. The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III*. The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma.

[Cloning and physical mapping of the ADE1 gene of the yeast Saccharomyces cerevisiae]. / Klonirovanie i fizicheskoe kartirovanie gena ADE1 drozhzhei Saccharomyces cerevisiae.

ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.

DNA supercoiling by core histone fractions and protamine.

The nuclear extract isolated from late Drosophila melanogaster embryos has Mg2+-dependent DNA topoisomerase 1 and nuclease activities. The extract facilitates the closed circular duplex DNA supercoiling in the presence of calf histone fractions H2A, H2B, H3 and H4, and fish protamine but not HI histone.

[Isolation, some properties and heterogeneous nature of DNA-topoisomerase (relaxing enzyme) from Ehrlich ascites carcinoma cells]. / Vydelenie, nekotorye svoistva i priroda geterogennosti DNK-topoizomerazy (relaksiruiushchego fermenta) iz kletok astsitnoi kartsinomy Erlikha.

DNA-topoisomerase catalyzing the conversion of a superhelical circular covalently closed DNA molecule into a super-helix free circular molecule, was isolated from mouse Ehrlich ascites carcinoma cells and purified 209-fold. The optimal conditions for the action and stability of the enzyme were elaborated. Using polyacrylamide gel electrophoresis under non-denaturating conditions as well as in the presence of Na-DS the heterogeneity of purified DNA-topoisomerase was established. This heterogeneity implies the presence of three active forms of the enzyme with Mr of 97 000, 81 000 and 69 000, respectively. Using one-dimensional fingerprint method and limited proteolysis with Staphylococcus aureus protease, it was demonstrated that the low molecular weight enzyme forms are products of limited proteolysis of the highest molecular weight form of DNA-topoisomerase.

[Complexes of histone H1 with supercoiled DNA]. / Issledovanie kompleksov gistona H1 s superspiral'noi DNK.

The action of DNA topoisomerase I on the complexes of supercoiled DNA (DNA I) with histone H1 and other histones was studied at different ionic conditions and histone/DNA ratios. In the presence of 0.15 M NaCl at histone H1/DNA ratios lower than 0.25 (w/w) the relaxation of DNA I is not inhibited. Raising of H1/DNA I ratio up to 0.7 is followed by significant inhibition of DNA I relaxation. At fixed H1/DNA I ratio maximal inhibition is detected at 0.25 M NaCl. At NaCl concentrations lower than 0.1 M and higher than 0.3 M increasing of DNA I relaxation in the presence of H1 was observed. Electron microscopic studies show that increase of ionic strength or H1/DNA I ratio causes more dense packing of DNA molecules in the H1.DNA complexes. Changes in the structure of complexes agree with the increase of DNA I relaxation inhibition in these conditions. DNA I relaxation inhibition by H1 is drastically decreased by iodination of tyrosine 72 residue in the globular part of H1 molecule. Individual core histones inhibit DNA I relaxation at much higher histone/DNA ratios and show different dependence of inhibition on ionic strength. The results are discussed in terms of the possible role of H1 in chromatin condensation-decondensation.

[Drosophila melanogaster embryo factor capable of supercoiling circular covalently closed DNA in the presence of core histones H2a, H2b, H3, H4 or protamine]. / Superspiralizatsiia kol'tsevoi kovalentno zamknytoi DNK v prisutstvii gistonov H2, H2b, H3, H4 ili protamina i ekstrakta iz embrionov Drosophila melanogaster.

The Mg2+-dependent factor capable of introducing the superhelical turns into circular closed DNA in the presence of core histones H2a, H2b, H3, H4 or protamine in solution at physiological conditions has been isolated from Drosophila melanogaster embryos. The factor does not work with histone H1, cytochrome c, poly-L-lysine or tetralysine. It is assumed that the factor is responsible for folding of core histones into nucleosomes.

[Two simple methods for isolation of DNA from various sources using cetavlon]. / Dva prostykh metoda vydeleniia DNK iz razlichnykh istochnikov s primeneniem tsetavlona.

Two general methods for the isolation of DNA from various sources based on the use of cetyltrimethylammonium bromide (cetavlon, CTA-Br) are described. Cetavlon is a strong cationic detergent precipitating DNA from diluted salt solutions. Cells are lysed and cellular components are dissolved in the presence of cetavlon, 5 M urea, 0.1 M EDTA and 2 M NaCl (KCl). In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the first method pure DNA is precipitated in the form of CTA-salt by direct dilution of the lysate to bring the concentration of NaCl (KCl) down to 0.5 M after the removal of the main part of proteins by deproteinization with chloroform. In the second method DNA is purified on the hydroxyapatite column after cell lysis and the removal of cell debris by centrifugation. Both methods are suitable for rapid isolation of pure DNA from various sources with recovery about 80% and average molecular weight 20-10(6) and higher without use of ribonuclease, pronase and amylase.