Betty L. McConaughy

The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin

The optimum monovalent cation concentration (Na+ or K+) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150-200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalent cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking -closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.

Related base sequences in the DNA of simple and complex organisms. VI. The extent of base sequence divergence among the DNAs of various rodents

The magnitude of differences which occur in the base sequence of DNAs from related animals was investigated through studies of four rodent DNAs. Fractionation on preparative CsCl density gradients and detailed examination of thermal denaturation profiles revealed the existence of several distinguishable components comprising the main band in addition to satellite bands. No difference was apparent in the distribution of unique and partially redundant sequences among these main band components, or in the apparent rate at which base substitution has occurred in evolution. Comparisons of sequence were also made by annealing reactions in solution at high concentration for extended periods with either labeled total mouse or rat DNA, or labeled unique sequences of mouse DNA. These experiments permitted assessment of the absolute extent of cross-reaction, as well as the extent of sequence divergence which had occurred between the two groups of interacting DNA molecules. Some implications of the apparent rapidity of nucleotide substitution in DNA are discussed.

Related base sequences in the DNA of simple and complex organisms. I. DNA/DNA duplex formation and the incidence of partially related base sequences in DNA

A comparative study has been made of the reactions of bacteriophage T4, B. subtilis,and mouse DNA with homologous DNA trapped on a membrane filter. The variation of reaction rate with temperature provides information as to the number of unique and partially related base sequences. The thermal stability of the duplexes formed at various temperatures has been used as a criterion of the extent of base pairing. These studies suggest that no closely related base sequences exist within the T4 genome. Similar results were obtained with Bacillus subtilisDNA although the data suggest the possibility of some very distant intragenome homologies. In contrast, mouse DNA contains so many related base sequences that the predominant reaction product is always one containing partially complementary strands. These results suggest that gene duplication has been a major mechanism operative in the increase of DNA content of metazoans through evolution. The relationship between the existence of partial base sequence redundancies and the mechanism of recombination is also discussed.

The interaction of oligodeoxynucleotides with denatured DNA

Abstract The stability and specificity of the interaction of oligodeoxynucleotides with denatured DNA was studied by incubating the mixture and exposing the reaction product to increasing temperatures. Oligomers of known chain length were prepared by partial depurination of labeled DNA, chromatographic fractionation of the resulting mixture and assay of terminal phosphate with alkaline phosphatase. As the size of the oligomers is increased, the extent of reaction with homologous DNA and the stability of the products increase. With chain lengths of 14 or less the interaction is not specific for homologous DNA. Clear specificity is demonstrable with larger fragments and the interaction with homologous DNA is much more stable to increasing temperature. Intermediate levels of partial homology are also apparent in the reaction between oligonucleotides and the DNA of a related organism.

Evolutionary complementation for polymerase II CTD function

The C‐terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle. The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms. Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all. The functional and evolutionary implications of this variation among RPB1 C‐termini is largely unexplored. We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild‐type CTD. The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad. Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress. These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD. Copyright

[8] Multiple forms of rat liver type I topoisomerase

Publisher Summary The type I topoisomerase from rat liver nuclei was originally described as a single-subunit enzyme with a molecular weight of 66,000. In subsequent experiments the addition of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) altered the elution profile from phosphocellulose and resulted in the isolation of higher molecular weight forms of the enzyme. The purification of the rat liver topoisomerase is re-examined in the presence of PMSF. Under these conditions one can identify an additional two, possibly three, forms of the topoisomerase that are separable by chromatography on phosphocellulose. This chapter describes the assay and partial characterization of these rat liver type I topoisomerases. In addition, the topoisomerase prepared from rat liver nuclei in the absence of PMSF appears to be adequate and, unlike the larger forms described here, can easily be obtained as a stable, homogeneous species in a reasonable yield.

Related base sequences in the DNA of simple and complex organisms. V. The specificity of interactions between oligonucleotides and denatured DNA

A mixture of differentially labeled mouse and Bacillus subtilis DNA was used as a source of oligodeoxynucleotides of chain lengths from 15 to 40 nucleotides. The extent of interaction of these oligonucleotides with homologous or heterologous DNA bound to membrane filters was measured. The specificity of such interactions increases with chain length and with the incubation temperature. The thermal stability of the complexes is a function of chain length. Homologous oligonucleotide/DNA duplexes of B. subtilis are more stable than those of mouse of corresponding size, consistent with the incidence of partially related base sequences in mouse DNA. Oligonucleotides in this size range are also able to recognize partially complementary base sequences in the DNA of closely related organisms. This approach shows promise as a means of obtaining quantitative estimates of base sequence divergence between the DNAs of related organisms.

Sulfur-containing derivatives of proteins

Abstract A number of new anhydrides have been prepared which contain disulfide, thioether and protected mercaptan groups. These include the following compounds: thiodiacetic anhydride, anhydride of dithiodiacetic acid, (carboxymethylmercapto)-succinic anhydride, ( β-N,N- dimethylaminoethyl )mercaptosuccinic anhydride, S- acetylthioitamalic anhydride. These have been found to react readily with bovine serum albumin and with gelatin and from 3–60 groups have been introduced per 100 000 g of protein. It should thus be possible to study the effects of various types of sulfur groups of the configuration and interactions of protein molecules.