A kinetic hairpin transfer model for parvoviral DNA replication

The DNAs encapsidated by parvoviruses show distinctly different patterns with respect to the ratio of plus-to-minus strands and sequence heterogeneity at the ends. A kinetic model, based on differential rates of hairpin transfer at 3' termini, is described and shown to account for all known parvoviral DNA distributions.     

Unique hairpin structures occurring at the replication origin of phage G4 DNA

Recently we reported that a DNA fragment, GCCAAAGC, forms an extraordinarily stable hairpin structure with two G-C pairs at the terminus and A-A-A stacked structure. The sequence is present at the replication origin of bacteriophage G4 ssDNA, and so on. Several kinds of possible hairpin structures, corresponding to the replication origin of phage G4, were synthesized and their secondary structures were examined. It was found that the fragments are able to form interconvertible hairpin structures depending on the length of the base-paired regions. The hairpin structure consisting of GCGAAAGC was not digested by the exonuclease activity of T4 DNA polymerase and it was stable enough to be only minimally bound by a single-stranded DNA binding protein.     

A double-loop model for the replication of eukaryotic DNA

Coordinated DNA synthesis of both strands at the replication fork by a fixed 'replisome' may cause dynamic and topological problems. Based upon known properties of DNA helicase, DNA primase and DNA topoisomerases, and on novel properties of DNA polymerases and DNA ligase, we propose a 'double-loop' model for the replication of eukaryotic DNA that could minimize such problems.     

DNA replication in Escherichia coli: physical and kinetic studies of the replication point

DNA replication in Escherichia coli was followed using analytical composite agarose-acrylamide gel electrophoresis of native and denatured DNA samples from cells that had been pulse-labeled in vivo. The results obtained with samples of native DNA, combined with the different sensitivities of the various electrophoretic fractions of native DNA to enzymatic attack, led us to conclude that a significant amount of single-strandedness exists in the region of the replication site. Data obtained from kinetic analyses of the labeling of both the high molecular weight DNA and the fragments formed on denaturation of native DNA samples were consistent with the Okazaki model (Okazaki et al., 1968a,b) of DNA replication. Furthermore, the data indicated that eight to ten precursor fragments are present per fork during bacterial growth at both 20 and 37 °C, under the conditions used in this study.     

Interconvertible hairpin structure found in the replication origin of phage G4 single-stranded DNA

We found a synthetic GCGAAAGC fragment with a mobility greater than that of other oligodeoxyribonucleotides in gel electrophoresis to take on a stable hairpin structure possessing two terminal G-C base pairs. The GCGAAAGC sequence is also found in the replication origin of phage G4 single-stranded DNA, but the hairpin structure originally proposed differs from that of the GCGAAAGC fragment we have studied. Possibility of rearrangement of the secondary structure in the replication origin of phage G4 was examined in relation to its replication initiation mechanism.     

Distance-dependent photoinduced electron transfer in synthetic single-strand and hairpin DNA

 The singlet state of stilbene-4,4′-dicarboxamide can serve as a fluorescent probe of both DNA conformation and electron transfer. Covalent incorporation of the stilbene-dicarboxamide into DNA structures with restricted conformational mobility results in inhibition of stilbene isomerization and an increase in its fluorescence quantum yield and lifetime. The fluorescence of stilbenedicarboxamide is selectively quenched by proximate guanine, but not by the three other DNA nucleobases. Selective quenching occurs via an electron transfer mechanism in which stilbene serves as the electron acceptor and guanine as the electron donor. Kinetic analysis of the distance dependence of electron transfer in stilbene-bridged hairpins suggests that duplex DNA is more effective than proteins as a medium for electron transfer, but that it does not function as a molecular wire. Received, accepted: 5 January 1998     

Initiation of adenovirus DNA replication does not occur via a hairpin mechanism.

Models have been proposed suggesting that initiation of adenovirus DNA replication might occur via a hairpin mechanism. A consequence of the models is a covalent linkage of progeny and parental DNA in newly completed molecules. Analysis of mature molecules from KB cells infected with adenovirus type 5 indicates that, if a hairpin mechanism is involved, the length of the hairpin must be shorter than 50 basepairs long. Recent nucleotide sequence analysis of the termini of adenovirus type 5 DNA (P.H.Steenbergh et al. (1977) Nucl. Acids Res. 4, 4371-4389) has shown that a hairpin of this size does not exist and that therefore a hairpin mechanism is unlikely.     

Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication.

The nucleotide sequences of the terminal regions of monomer replicative form DNA, a pivotal intermediate species in the replication of minute virus of mice, were determined. The left (3') terminus had a unique sequence on both strands and in both 3'-hairpin configurations. In contrast, the right (5') terminus was sequence heterogeneous and extended an additional 18 base pairs beyond that expected from the known sequence of the virion DNA. These data unambiguously establish the sequence complexity at the termini of both the single-stranded viral genome and the pool of replicative DNA. A comparison of the combined sequence information leads us to propose a modified rolling hairpin model for the replication of autonomous parvoviruses which is compatible with all available data.     

AcMNPV as a model for baculovirus DNA replication

Baculoviruses were first identified as insect-specific pathogens, and it was this specificity that lead to their use as safe, target specific biological pesticides. For the past 30 years, AcMNPV has served as the subject of intense basic molecular research into the baculovirus infectious cycle including the interaction of the virus with a continuous insect cell line derived from Spodoptera frugiperda. The studies on baculoviruese have led to an in-depth understanding of the physical organization of the viral genomes including many complete genomic sequences, the time course of gene expression, and the application of this basic research to the use of baculoviruses not only as insecticides, but also as a universal eukaryotic protein expression system, and a potential vector in gene therapy. A great deal has also been discovered about the viral genes required for the replication of the baculovirus genome, while much remains to be learned about the mechanism of viral DNA replication. This report outlines the current knowledge of the factors involved in baculovirus DNA replication, using data on AcMNPV as a model for most members of the Baculoviridae.     

General kinetic model for protein-mediated phospholipid transfer between membranes

Phospholipid transfer protein catalyzes the transfer of phospholipids between bilayer membranes. A general model is developed for describing the kinetics of this process. While previous models derive detailed expressions only for the initial rate of transfer from donor to acceptor membranes, this model takes into account donor-to-donor, acceptor-to-acceptor, and acceptor-to-donor transfers, in addition to the usual donor-to-acceptor transfer. The apparent rate of transfer along any of these specific routes is given as the product of the total rate of transfer (the sum of the rates of transfer along all four routes) and a probability function uniquely defined for each route. The model explains adequately the effects of membrane concentration on phospholipid transfer activity as well as the consequences of varying membrane surface charge and size. Using bovine liver phosphatidylcholine transfer protein, the model is applied to the kinetic analysis of phosphatidylcholine transfer between two populations of small unilamellar vesicles. Rates of protein-catalyzed phosphatidylcholine transfer between vesicles with identical phosphatidic acid content (2 or 6 mol%) are determined experimentally as a function of total vesicle concentration to calculate apparent dissociation constants and maximum rates of transfer; apparent rates of transfer between various combinations of vesicles containing 2 or 6 mol% phosphatidic acid are then deduced from the derived velocity expression. Reasonably good agreement is seen between theoretical apparent rate-vesicle concentration relationships and those measured experimentally. The results support the general treatment of the kinetics of protein-mediated phospholipid transfer and permit an estimation of useful kinetic parameters.