Human immunodeficiency virus type 1 central DNA flap: dynamic terminal product of plus-strand displacement dna synthesis catalyzed by reverse transcriptase assisted by nucleocapsid protein.

To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the proviral DNA generating a 99-nucleotide DNA flap (6). This step, catalyzed by reverse transcriptase (RT), is defined as a discrete strand displacement (SD) synthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Using recombinant HIV-1 RT and a circular single-stranded DNA template harboring the cPPT-CTS sequence, we have developed an SD synthesis-directed in vitro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), the main accessory protein of the reverse transcription complex. A full-length HIV-1 central DNA flap was then carried out in vitro. Its synthesis appears faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (gp32). Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and efficient 3'-5' branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the NCp chaperone activity. The biological implications of the RT-directed NCp-assisted flap synthesis are discussed within the context of reverse transcription complexes, assembly of the preintegration complexes, and nuclear import of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.

HIV-1 reverse transcription. A termination step at the center of the genome.

During HIV-1 reverse transcription, the plus-strand of viral DNA is synthesized as two discrete segments. We show here that synthesis of the upstream segment terminates at the center of the genome after an 88 or 98 nucleotide strand displacement of the downstream segment, initiated at the central polypurine tract. Thus, the final structure of unintegrated linear HIV-1 DNA includes a central plus-strand overlap. In vitro reconstitution using only purified reverse transcriptase with appropriate DNA hybrids gave rise to efficient and accurate termination, which was dramatically amplified in the context of strand displacement. Mutation of the sequence immediately upstream of the termination sites almost completely abolished termination both in infected cells and in vitro. This mutation profoundly impaired replication of HIV-1. We conclude that proper central plus-strand termination, mediated by a novel cis-active termination sequence, is a key step in HIV-1 replication.

"In situ" characterization of GnRH receptors: use of two radioimagers and comparison with quantitative autoradiography.

New radioimagers, the HRRI (high resolution radioimager) and the Phosphorimager (phosphor screen : PS), apt to display more ample linear dose-response scale than radio-sensitive films, were tested in comparison with quantitative autoradiography (QA). GnRH receptor saturation experiments were achieved on tissue sections (rat pituitary, rat brain, human ovary) with a iodinate GnRH agonist (125I-[D-Ala6,Des-Gly10]-LH-RH Ethylamide) for determination of affinity constant (Kd). In rat pituitary, comparable results were obtained with the 3 methods (Kd: 0.4 to 0.6 nM). Discrepancies occurred in the hippocampus and in the granulosa cell layer of the preovulatory follicle, due to low resolutive (PS) or short linear dose-response (films) performances. In the hippocampus GnRH receptor affinity was under-estimated with PS (Kd: 2.3 vs 0.5 and 0.6 nM for QA and HRRI respectively). In the follicular granulosa cell layer it was over-estimated by QA (0.5 vs 50 nM for the HRRI), while PS did not allow resolution of this thin cell layer. In conclusion, the HRRI is a very powerful tool for the quantification of in situ radioligand binding (binding sites study and in situ hybridization) in very discrete areas.

Mg2+, Asp-, and Glu--effects in the processive and distributive DNA relaxation catalyzed by a eukaryotic topoisomerase.

The salt requirement for the catalysis of DNA relaxation carried out by a eukaryotic DNA topoisomerase I from Candida was reexamined with plasmid pBR322 DNA. Two levels of analysis were considered: the initial velocity of the overall reaction and the mode of this reaction (processivity vs distributivity). When looking at the monovalent salts from the first level, the replacement of Cl- by Glu- or Asp- greatly enhanced the salt range over which the enzyme was active. Moreover, the initial velocity reached an optimal value for a higher salt concentration in this case. For the cationic counterpart, K+ was a little more effective than Na+ and much more so than NH4+. Addition of 4 mM magnesium chloride affected both the range and the optimum of the initial velocity differentially, depending upon the monovalent salt, but with a general stimulating tendency. On the other hand, when the Mg2+ salt was varied, substitution of chloride by aspartate enhanced the optimum of the initial velocity for a fixed KCl concentration. In addition, magnesium aspartate (MgAsp2) and magnesium glutamate (MgGlu2) allowed the reaction to occur even without monovalent salt and over an extended range. Magnesium was also shown to directly interact with the general catalysis (Kd = 2.5 mM). From the second level of analysis, the presence of Mg2+ (except with NH4Glu), the substitution of Cl- by Glu- or Asp-, and a lower monovalent salt concentration than that used for the velocity optimum were required to promote the processive mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Reverse gyrase binding to DNA alters the double helix structure and produces single-strand cleavage in the absence of ATP.

Stoichiometric amounts of pure reverse gyrase, a type I topoisomerase from the archaebacterium Sulfolobus acidocaldarius were incubated at 75 degrees C with circular DNA containing a single-chain scission. After covalent closure by a thermophilic ligase and removal of bound protein molecules, negatively supercoiled DNA was produced. This finding, obtained in the absence of ATP, contrasts with the ATP-dependent positive supercoiling catalyzed by reverse gyrase and is interpreted as the result of enzyme binding to DNA at high temperature. Another consequence of reverse gyrase stoichiometric binding to DNA is the formation of a cleavable complex which results in the production of single-strand breaks in the presence of detergent. Like eubacterial type I topoisomerase (protein omega), reverse gyrase is tightly attached to the 5' termini of the cleaved DNA. In the light of these results, a comparison is tentatively made between reverse gyrase and the eubacterial type I (omega) and type II (gyrase) topoisomerases.

Reverse gyrase of Sulfolobus: purification to homogeneity and characterization.

By using hydrophobic interaction as the first chromatographic stage, we purified to homogeneity reverse gyrase, an ATP-dependent DNA topoisomerase I, isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldrius. This procedure allowed quick and complete separation of reverse gyrase from nucleases and DNA binding proteins present in Sulfolobus. The final product was revealed, by SDS-PAGE, as a unique band with an apparent molecular mass of 128 kDa, and the amino acid composition was determined. Western blotting experiments with antibodies raised against reverse gyrase indicate that no proteolysis occurred during the purification course. Gel filtration and sedimentation data gave a Stokes radius of 42 A and a sedimentation coefficient of 5.7 S, suggesting a monomeric structure for the native enzyme which was confirmed by electron microscopy. Finally, pure reverse gyrase in a monomeric state was still able to promote positive supercoiling of the DNA.

Topoisomerase inhibitors induce irreversible fragmentation of replicated DNA in concanavalin A stimulated splenocytes.

Etoposide, a nonintercalative antitumor drug, is known to inhibit topoisomerase II. Its effects have been tested in concanavalin A stimulated splenocytes, a system of cell proliferation in which topoisomerase II is induced. The primary effect of etoposide was a strong inhibition of DNA synthesis and the production of reversible DNA breaks, presumably associated with topoisomerase II. However, prolonged (20 h) contact with the drug resulted in a secondary fragmentation by irreversible double-strand breaks that yielded unusually small DNA fragments. Surprisingly, the same effect was obtained with novobiocin, which does not produce topoisomerase II associated DNA breaks. Moreover, long-term treatment with camptothecin, a specific inhibitor of topoisomerase I which is known to induce single-strand breaks in vitro and in vivo, also produced double-strand breaks and DNA fragmentation into small pieces. These findings suggest that prolonged treatment of proliferating splenocytes by etoposide and other topoisomerase inhibitors induced DNA fragmentation by a mechanism that does not directly involve topoisomerases.

High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius.

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=

ATP-dependent DNA topoisomerase from the archaebacterium Sulfolobus acidocaldarius. Relaxation of supercoiled DNA at high temperature.

A topoisomerase, able to relax negatively supercoiled DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. Relaxation was fully efficient in vitro between 70 degrees C and 80 degrees C and was dependent on the presence of ATP and magnesium ions. The enzyme did not exhibit gyrase-like activity and was poorly sensitive to gyrase inhibitors. These properties are reminiscent of eukaryotic type II topoisomerases. However, the enzyme was unable to relax positively supercoiled DNA. This thermophilic enzyme may be used in a variety of ways to study the structure and stability of DNA at high temperature.

DNA topoisomerase activities in concanavalin A-stimulated lymphocytes.

Topoisomerase activities have been measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.

DNA topoisomerases from rat liver: physiological variations.

Besides the nicking-closing (topoisomerase I) activity, an ATP-dependent DNA topoisomerase is present in rat liver nuclei. The enzyme, partially purified, is able to catenate in vitro closed DNA circles in a magnesium-dependent, ATP-dependent, histone H1-dependent reaction, and to decatenate in vitro kinetoplast DNA networks to yield free minicircles in a magnesium-dependent and ATP-dependent reaction. It is largely similar to other eukaryotic type II topoisomerases in its requirements, and presumably belongs to this class of enzymes. Type I and type II activities were measured in rat liver nuclei as a function of regenerating time after partial hepatectomy: type I activity was not significantly changed during this process. In contrast, type II activity was considerably increased, suggesting a possible involvement of the enzyme in DNA replication.