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M Abdel-Monem

Publications and source records attributed to M Abdel-Monem.

15 recordsLinked to original sources
PubMed1992

Termination complex in Escherichia coli inhibits SV40 DNA replication in vitro by impeding the action of T antigen helicase.

DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases.

Antigens, Polyomavirus Transforming↗
PubMed1986

Purification of Escherichia coli DNA helicase I from plasmid-transformed cells.

DNA helicase I was purified in large quantity from Escherichia coli cells harboring a plasmid that carries the gene encoding helicase I--the traI gene of the F sex factor--cloned in a high copy number vector. Electron microscopic studies on the purified material reveal new properties of the enzyme protein.

DNA Helicases↗
PubMed1985

Effect of DNA polymerase I and DNA helicase II on the turnover rate of UvrABC excision nuclease.

UvrABC excision nuclease (UvrA, UvrB, and UvrC proteins) of Escherichia coli removes nucleotide mono- and diadducts from DNA in the form of oligonucleotides 12 or 13 bases long. We find that the purified enzyme dissociates from DNA very slowly, if at all, in the absence of other proteins implicated in excision repair. Addition of DNA polymerase I and helicase II (UvrD protein) to the reaction mixture stimulates the turnover rate of the excision nuclease to a level comparable to that observed in vivo.

Adenosine Triphosphatases↗
PubMed1983

Identification of Escherichia coli DNA helicase I as the traI gene product of the F sex factor.

Active DNA helicase I (Mr 180,000) can be isolated from Escherichia coli F+ strains but not F- strains. The transfer of the F sex factor to F- strains by conjugation permits the purification of the enzyme from the transconjugant strains. We conclude from this that helicase I is coded for by a portion of the F factor. Results also obtained by using recombinant plasmids carrying different DNA fragments of the F factor transfer region suggest that DNA helicase I is identical to the product of traI, one of the transfer genes of the F factor.

Base Composition↗
PubMed1982

DNA synthesis at a fork in the presence of DNA helicases.

In a mixture of Escherichia coli DNA polymerase III holoenzyme, single-strand-binding protein, artificially forked lambda bacteriophage DNA with primer annealed to the leading side of the fork, dNTPs and ATP, DNA synthesis is enhanced by helicase II, less so by helicases, I, III or rep protein of E. coli or T4 phage helicase. The effect of helicase II depends on ATP, it is enhanced by helicase III, and it is not observed using DNA polymerase I or T4 DNA polymerase. In the absence of dNTPs helicase II is less active than helicase I or T4 helicase in unwinding the forked DNA. We believe that helicase II both shifts the forks and stimulates DNA polymerase III. The results support the conclusion derived from previous studies that helicase II is part of the DNA-synthesizing system of E. coli.

Adenosine Triphosphatases↗
PubMed1980

Studies on the functions of DNA helicase I and DNA helicase II of Escherichia coli.

Inactivating antibodies raised against DNA helicase I and DNA helicase II were applied to Escherichia coli DNA-replicating systems. Antibody against DNA helicase II was found to inhibit the replication of E. coli DNA, lambda phage DNA (during early and late phases), and ColE1 plasmid DNA during the elongation step. The antibody did not inhibit the replication of fd replicative form (RF) DNA, the unwinding of which is known to depend on the rep protein. Antibody against DNA helicase I failed to inhibit any of the replication processes. The replication of E. coli DNA, lambda-DNA, and ColE1 DNA is known to be initiated in a closed circle, in contrast to fd RF which is known to be initiated in a nicked circle. In conjunction with data given in the literature, our results suggest that replicative unwinding is carried out by DNA helicase II or rep protein, depending on the mechanism by which DNA replication is initiated. The concentration of DNA helicase II in E. coli, as determined by immunological methods, is 5000 to 8000 copies/cell; that of DNA helicase I is 500 to 700.

Adenosine Triphosphatases↗
PubMed1979

DNA helicases.

In summary, we postulate that DNA unwinding and ATP dephosphorylation are coupled in different ways, depending on whether the fibrous ATPase or one of the globular ATPases provides the catalytic agent. Unanswered is the question of whether there is stoichiometry of ATP utilization during the unwinding of a duplex, and unsolved is the role of the individual enzyme in the cell.

Adenosine Triphosphatases↗
PubMed1977

DNA unwinding enzyme II of Escherichia coli. 1. Purification and characterization of the ATPase activity.

A DNA-stimulated ATP-gamma-phosphohydrolase of molecular weight 75000 was purified from Escherichia coli cells. The ATPase, a globular molecule (identical probably with an ATPase described previously by Richet and Kohiyama in 1976) shows specificity for adenine nucleotides, it prefers single-stranded DNA as the cofactor, it exhibits a complicated mode of response to variations of the cofacter concentration and it is devoid of nuclease activity. Preparations derived from rep3 mutant cells yield widely varying amounts of an apparently normal ATPase.

Adenosine Triphosphatases↗
PubMed1977

DNA unwinding enzyme II of Escherichia coli. 2. Characterization of the DNA unwinding activity.

The DNA-stimulated 75000-Mr ATPase described in the preceding paper is shown to be a further catalytic DNA unwinding principle (DNA unwinding enzyme II) made in Escherichia coli cells (the first being the 180000-Mr ATPase of the cells: DNA unwinding enzyme I). Unwinding depends strictly, on the supply of ATP. It occurs only under conditions permitting ATP dephosphorylation and it proceeds as long as enzyme molecules are permitted to enter the enzyme - DNA complex. The enzyme binds specifically to single-stranded DNA yielding a complex of only limited stability. These results are interpreted in terms of a distributive mode of action of the enzyme. It is argued that chain separation starts near a single-stranded DNA region and that, forced by continued adsorption of enzyme molecules to the DNA, it develops along the duplex. This mechanism is different from that deduced previously for DNA unwinding enzyme I. Complicated results were obtained using ATPase prepared from rep3 mutant cells.

Adenosine Triphosphatases↗
PubMed1976

Enzymic unwinding of DNA. 1. Purification and characterization of a DNA-dependent ATPase from Escherichia coli.

Evidence from various sources in the literature suggests that, in connection with DNA, ATP dephosphorylation can be used to provide energy for mechanical effects. Starting from this concept we have studied a novel DNA-dependent ATPase purified to 90% homogeneity from Escherichia coli. The enzyme has a peptide weight near 180 000 and, in high salt, is a monomeric, probably highly anisometric molecule. In salt-free buffer, where the ATPase activity is highest, the enzyme forms aggregates. ATP is the preferred substrate (Km 0.27 mM) and dephosphorylated at the gamma-position at a maximal rate near 10(4) molecules per enzyme monomer per min at 35 degrees C. A requirement for divalent cation is best satisfied by Mg2+ or Ca2+ and the requirement for DNA best by the single-stranded, circular DNA of phages phiX174 (Km 62 nM nucleotide) and fd indicating that the enzyme recognizes internal DNA regions. When saturated with E. coli DNA unwinding protein phiX DNA is not accepted but, once in contact with the DNA, the enzyme is little inhibited by unwinding protein. Apparently the unwinding protein interferes preferentially with the recognition of DNA. The enzyme does not detectably cleave DNA, and for this and genetic reasons is not identical with the recBC ATPase or the K12 restriction ATPase of the extracted cells. The enzyme is probably not identical either with the dnaB-product-associated ATPase or the ATPase activity found in DNA polymerase III holoenzyme under appropriate conditions, and it is certainly not identical with a DNA-dependent ATPase of molecular weight 69 000 from E. coli which has recently been purified. Attempts to ascribe the enzyme to other genes, including recA, lex and rep, have failed.

Adenosine Triphosphatases↗
PubMed1976

Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme.

The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA, RNA hybrid duplexes and DNA-DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.

Adenosine Triphosphatases↗