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L F Liu

Publications and source records attributed to L F Liu.

173 records · Page 10Linked to original sources
PubMed1981

Knotted DNA from bacteriophage capsids.

The majority of the DNA prepared from tailless capsids of bacteriophage P2 by the phenol extraction procedure consists of monomeric rings that have their cohesive ends joined. Electron microscopic and ultracentrifugal studies indicate that these molecules have a complex structure that is topologically knotted; they have a more compact appearance and a higher sedimentation coefficient when compared with regular nicked P2 DNA rings. Linearization of these rings by thermal dissociation or repair of the cohesive ends by DNA polymerase I in the presence of all four deoxynucleoside triphosphates gives molecules that are indistinguishable from normal P2 DNA that has been similarly treated. The knotted nature of the majority of P2 head DNA is further supported by analyzing the products when these molecules are treated with ligase and the ligase-treated molecules are subsequently nicked randomly with DNase I. The data are consistent with the notion that, if such a molecule is first converted to a form that contains only one single-chain scission per molecule, strand separation gives a linear strand and a highly knotted single-stranded ring. The results suggest that the DNA packaged in tailless P2 capsids is arranged in a way that leads to the formation of a complex knot when the ends join. In an intact phage particle, the anchoring of one terminus of the DNA to the head-proximal end of the tail [Chattoraj, D. K. & Inman, R. B. (1974) J. Mol. Biol. 87, 11-22] presumably diminishes or prevents this kind of joining. The novel knotted DNA can be used to assay type II DNA topoisomerases that break and rejoin DNA in a double-stranded fashion.

Capsid↗
PubMed1980

Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break.

The T4 DNA topoisomerase is a recently discovered multisubunit protein that appears to have an essential role in the initiation of T4 bacteriophage DND replication. Treatment of double-stranded circular DNA with large amounts of this topoisomerase in the absence of ATP yields new DNA species which are knotted topological isomers of the double-stranded DNA circle. These knotted DNA circles, whether covalently closed or nicked, are converted to unknotted circles by treatment with trace amounts of the T4 topoisomerase in the presence of ATP. Very similar ATP-dependent enzyme activities capable of unknotting DNA are present in extracts of Drosophila eggs. Xenopus laevis eggs and mammalian tissue culture cells. The procaryotic enzyme, DNA gyrase, is also capable of unknotting DNA. We propose that these unknotting enzymes constitute a new general class of DNA topoisomerases (type II DNA topoisomerases). These enzymes must act via mechanisms that involve the concerted cleavage and rejoining of two opposite DNA strands, such that the DNA double helix is transiently broken. The passage of a second double-stranded DNA segment through this reversible double-strand break results in a variety of DNA topoisomerization reactions, including relaxation:super-coiling; knotting:unknotting and catenation:decatenation. In support of this type of mechanism, we demonstrate that the T4 DNA topoisomerase changes the linking number of a covalently closed double-stranded circular DNA molecule only by multiples of two. We discuss the possible roles of such enzymes in a variety of biological functions, along with their probable molecular mechanisms.

Animals↗
PubMed1979

Nonhistone proteins HMG1 and HMG2 unwind DNA double helix.

In a previous communication we have shown that both HMG1 and HMG2 nonhistone proteins change the DNA helical structure and the binding of HMG1 and HMG2 to DNA induces a net unwinding equivalent of DNA double helix (Javaherian, K., Liu, L. F. and Wang, J. C. (1978) Science, 199, 1345-1346). Employing melting absorption technique, we now show that in the presence of salt HMG1 and HMG2 destabilize DNA whereas in the absence of salt, they both stabilize DNA molecules. Consequently the folded structure of HMG must play an important role in melting DNA. Furthermore, by measuring topological winding number using competition unwinding experiments, we conclude that HMG1 has a higher affinity for a single-stranded DNA relative to double-stranded DNA. These results together suggest that HMG1 and HMG2 unwind DNA double helix by local denaturation of the DNA base pairs. The net unwinding angles have been measured to be 22 degrees and 26 degrees per molecule of HMG1 and HMG2 respectively.

Animals↗
PubMed1978

Interaction between DNA and Escherichia coli protein omega. Formation of a complex between single-stranded DNA and omega protein.

Escherichia coli omega protein is found to form a complex with single-stranded DNA. The complex is stable in buoyant CsCl or Cs2SO4 density gradients. Addition of Mg(II) to the concentrated salt solutions, however, leads to the dissociation of the complex, even in the presence of EDTA in molar excess over Mg(II). The dissociated omega retains its enzymatic activity; the DNA recovered from the dissociated complex is indistinguishable from the original DNA. Exposure of the complex to alkali results in the cleavage of the DNA. This cleavage generates a 3'-hydroxyl DNA terminus, and the omega protein is found linked to the 5'-terminus, presumably covalently. Pronase digestion of the complex results initially in the removal of approximately 30% of the protein. A significant fraction of the residual complex is still stable in concentrated salt solutions, and can be dissociated by Mg(II). Extensive digestion with pronase results in the removal of the protein and the cleavage of the DNA chain.

Bacterial Proteins↗
PubMed1978

Micrococcus luteus DNA gyrase: active components and a model for its supercoiling of DNA.

Two active components alpha and beta of micrococcus luteus DNA gyrase, of peptide weights of 115,000 and 97,000, respectively, have been purified. Each individual component exhibits little DNA gyrase activity; the ATP-dependent negative supercoiling of a covalently closed circular DNA duplex is catalyzed by a combination of the two. Covalent closure by Escherichia coli ligase of a circular DNA containing single-chain scissions, when carried out in the presence of a combination of the DNA gyrase components alpha and beta, gives a positively supercoiled DNA upon removal of the bound protein molecules. ATP was not present during the ligase treatment; therefore the positive supercoiling of DNA observed is a result of the binding of gyrase molecules, presumably as multi-subunit oligomers, during the ligation step. This is in contrast to the negative supercoiling of DNA catalyzed by gyrase in the presence of ATP. A model in which negative supercoiling of DNA is achieved by ATP-modulated repetitive wrapping of the DNA around gyrase is described. The model also suggests a plausible mode of action by which translocation of a DNA along its helix axis can be actively driven by an ATPase.

Adenosine Triphosphate↗
PubMed1975

On the degree of unwinding of the DNA helix by ethidium. II. Studies by electron microscopy.

When a negatively twisted covalently closed DNA is annealed with single-stranded fragments of the same DNA, under proper conditions a loop (or loops) may form by the disruption of a segment (or segments) of base pairs between the complementary strands of the covalently closed DNA, and the formation of base pairs between the strands of the covalently closed DNA and the single-stranded fragments. Since such a process involves essentially no net gain or loss of the number of base pairs, it is driven by the free energy favoring the reduction of the number of superhelical turns. If the fragments are sufficiently long or are present at a sufficiently hig concentration during annealing, the most stable product between a covalently closed DNA and the DNA fragments (under conditions favoring the formation of double-stranded DNA) is a looped molecule devoid of superhelical turns. The size of the looped region or regions, which can be measured by electron microscopy, provides a way to determine the degree of superhelicity of the covalently closed DNA in the absence of the fragments. When this is compared with the degree of superhelicity of the covalently closed DNA determined by titration with the intercalative dye ethidium, the unwinding angle of the DNA double helix due to the intercalation of an ethidium can be calculated. Such measurements were done on two samples of phage PM2 DNA with different extents of supercoiling. The results are in agreement with the value 26 degree obtained recently by alkaline titration of covalently closed PM2 DNA samples in CsC1 density gradients (Wange, J.C., (1974) J. Mol. Biol. 89, 783-801).

Bacteriophages↗
PubMedDate not supplied

Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells.

DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes. Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide. Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA. We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin. The formation of covalent complexes seems to be reversible on removal of teniposide. Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments. Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.

Adenocarcinoma↗