Heinz Schaller

The topology of DNA from the small filamentous bacteriophage fd.

Physically homogeneous preparations of DNA can be isolated from fd. This DNA is resistant to hydrolysis by exonuclease I. The kinetics of pancreatic DNase action on the DNA as followed by sedimentation and viscosity in suitable solvents show that a single hit (biological inactivation) leads to an expansion of the DNA structure but no change in molecular weight. The native DNA molecule is resistant to a variety of agents which destroy secondary structure. These results are taken to indicate that the DNA has a stable ring topology.

Deoxyribonucleic acid replication in vitro

Abstract We describe an in vitro system for DNA replication which uses a highly concentrated lysate of DNA polymerase I deficient Escherichia coli bacteria. The DNA synthesis observed in vitro proceeds over long periods of time, and the rates of total DNA synthesis and of chain, elongation are 10 to 20% of the in vivo rate. This in vitro DNA synthesis resembles in vivo replication in many aspects. It is semiconservative. Furthermore the DNA is synthesized in small pieces which become joined together upon prolonged incubation. Treatments which specifically inhibit in vivo replication affect the rate of in vitro synthesis. The rate is much reduced when the cells have been pretreated with ultraviolet light or mitomycin, when cells have been allowed to finish the replication cycle in the absence of further initiation, when synthesis takes place in the presence of nalidixic acid, or when synthesis takes place at non-permissive temperature in lysates of mutants that are temperature-sensitive with respect to DNA replication. DNA synthesis depends on the presence of soluble macromolecules other than DNA polymerase I or II. These molecules must be present at a concentration comparable to their in vivo concentration.

Structure of the DNA of bacteriophage fd: II. Isolation and characterization of a DNA fraction with double strand-like properties

Abstract By enzymic degradation with two single strand-specific nucleases, exonuclease I from Escherichia coli and endonuclease from Neurospora crassa, a double strand-like DNA core has been detected in the single-stranded DNA of bacteriophage fd. From the kinetics of exonuclease I action at 30 °C the resistant DNA core has been estimated to represent 2.5% of the total DNA, corresponding to 150 out of 6000 nucleotide residues. In preparative experiments about 1.5% of the total nucleotides in fd DNA were isolated after limited nuclease digestion as core DNA fragments with an average chain length of about 40 nucleotide residues. In addition to its resistance to degradation by single strand-specific nucleases, fd DNA core is characterized by a high G + C-content, a simple pyrimidine distribution, and double strand-like spectral properties. Fd DNA core chains rapidly recover their secondary structure following denaturation. The data are interpreted in terms of a secondary structure of a single polynucleotide chain folding back on itself.

Structure of the DNA of bacteriophage fd: I. Absence of non-phosphodiester linkages

Abstract When the DNA from bacteriophage fd is treated first with an endonuclease and then with an exonuolease, all molecules resistant to exonucleolytic attack are circular molecules which have not received an endonucleolytic cut. In linear molecules no evidence could be found for a covalent bond which could not be cleaved by exonuclease I. A similar result was obtained for bacteriophage φX174, The products of enzymic hydrolysis of highly 32 P-labelled fd DNA were analysed by two-dimensional thin-layer chromatography, an assay which could detect as little as a single phosphorus atom per DNA molecule. Only the four common deoxynucleotides were found after extensive action of pancreatic DNase and venom phosphodiesterase. From these data it is concluded that fd DNA and φX DNA are continuous polynucleotide chains.

A new approach to the sequence analysis of DNA

For the analysis of structure and function of DNA it would be of advantage, if short defined segments of the macromolecule could be copied in vitro. DNA polymerase I is able to initiate DNA synthesis at internal sites on a single-stranded DNA template, if oligonucleotides are offered as primers [ 1, 21. Recent experiments indicated that the initiation reaction may be sequence specific, since a high template specificity was found when oligonucleotides were used as primers which were long enough to anneal stably fo a template DNA [3,4]. In the present communication we show that DNA polymerase I can initiate DNA synthesis at a single site on a phage DNA template, if a unique base complementary oligonucleotide is offered as a primer. Fd DNA minus strands were used as template, the polypyrimidine tract C9Tll from the fd plus strand [S] as primer. Pulse-labelled reaction products of various size were characterized by fingerprinting techniques, which allowed to deduce the sequential arrangement of the polypyrimidine tracts in the nucleotide sequence following the 3’ terminus of the primer nucleotide.