Joel A. Huberman

The in vivo replication origin of the yeast 2μm plasmid

Abstract We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2μm plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 ± 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2μm plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA.

Stimulation of T4 bacteriophage DNA polymerase by the protein product of T4 gene 32

Abstract The protein product of T4 bacteriophage gene 32 (32-protein) is required for replication of T4 DNA in vivo . Alberts, Amodio, Jenkins, Butmann & Ferris (1968) have shown that this protein binds tightly and co-operatively to single-stranded DNA under physiological ionic conditions in vitro , and in so doing it holds the otherwise highly folded DNA chain in a much more extended conformation (Alberts & Frey, 1970). We have now tested the effect of 32-protein on the activity of T4 DNA polymerase, an enzyme which requires a single-stranded DNA template, and find that it stimulates the in vitro rate of polymerization on such templates 5- to 10-fold. The rate of stimulated synthesis is about 10% of that measured for movements in vivo of growing points. The 32-protein from a temperature-sensitive mutant in gene 32 ( ts P7) is also temperature-sensitive in the stimulation of T4 polymerase in vitro , suggesting that this stimulation is related to the intracellular function of 32-protein in DNA replication. The stimulation of T4 DNA polymerase by 32-protein is greatest at low temperature, high ionic strength and at a level sufficient to bind most or all of the template DNA present. These results suggest that 32-protein stimulates this polymerase by removing inhibitory secondary structure from the template DNA strand. The activity of DNA polymerase from Escherichia coli on single-stranded DNA templates is not significantly affected by 32-protein. In addition, in the absence of DNA, a weak complex is formed between 32-protein and T4 DNA polymerase, whereas no such complex is formed with E. coli DNA polymerase. Thus, some specific interaction between polymerase and 32-protein may also be essential for the stimulation of DNA synthesis observed.

Both DNA topoisomerases I and II relax 2 μm plasmid DNA in living yeast cells

Abstract Measurements at various temperatures of the linking number of yeast 2 μm plasmid DNA in wild-type cells and in cells bearing mutations in the DNA topoisomerase I and II genes show that bulk 2 μm plasmid minichromosomes are maintained in a relaxed state by the combined action of topoisomerases I and II. Bulk 2 μm minichromosomes are not under torsional stress in vivo and are not substrates for a putative gyrase-like topoisomerase.

Electron microscopy of DNA polymerase bound to DNA.

Abstract High-resolution mounting and shadowing techniques were applied to the electron microscopic visualization of Escherichia coli DNA polymerase, free and bound to DNA. The enzyme appeared to be roughly spherical with a diameter of about 65 A; dimeric forms, linked by mercury between the cysteine side chains, appeared as pairs of spheres, 130 A × 56 A. Enzyme molecules were observed at regular intervals along a DNA helix composed of about 20 polydeoxythymidylate chains aligned along a polydeoxyadenylate chain. This binding corresponds to attachment of the enzyme at the points of apposition of thymidylate chains, namely, nicks in the helical structure; little or no binding was observed along the length of unnicked phage λ DNA. Enzyme molecules were oriented both over and to the side of the DNA chain implying a shallow rather than a very deep groove for the area of attachment. With one molecule of a dimeric enzyme attached to DNA, the other member of the pair assumed a variety of juxtapositions suggesting considerable flexibility in the area of cross-linkage.