Lawrence A. Loeb

ZINC in DNA polymerases

Summary Homogeneous DNA polymerases from E. coli and sea urchins contain zinc in a proportion of approximately 2 and 4 gm atoms Zn/mole enzyme respectively. Specific inhibition of the enzyme by ortho -phenanthroline and lack of inhibition by meta -phenanthroline suggest that the bound zinc plays a functional role in the interaction of DNA with the polymerases.

Sea urchin nuclear DNA polymerase: II. Changing localization during early development

Abstract In sea urchin embryos, there is little change in the total DNA polymerase activity during early development. However, there is a progressive increase in the per cent of this activity which is associated with the nuclear fraction and at the same stage there is a decrease in the amount of activity in the soluble cytoplasmic fraction. By the hatching stage (about 300 cells/embryo) most of the DNA polymerase activity is found in the nucleus. The intracellular migration of polymerase from the cytoplasm to the nucleus is suggested as a mechanism to account for these results.

Metal activation of DNA synthesis

We have determined that Co2+, Ni2+ or Zn2+ may substitute for Mg2+ during DNA synthesis with E.coli DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co2+ or Mn2+ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.

DNA polymerase activity as an index of lymphocyte stimulation: studies in Down's syndrome

The ability of peripheral blood lymphocytes to respond to phytohemagglutinin (PHA) in vitro was studied in patients with Downs syndrome. The response was measured by the increase in DNA polymerase activity and the rate of incorporation of tritiated thymidine by the cultured lymphocytes. These activities were significantly lower in PHA-stimulated lymphocytes from patients with Downs syndrome compared with age- and sex-matched, mentally retarded patients without Downs syndrome from the same institution and the normal healthy volunteers. The impairment in response to PHA does not seem to be related to the presence of Australia antigen in patients with Downs syndrome or to institutionalization itself. In contrast to DNA polymerase activity and thymidine-(3)H uptake, there was no significant difference in the percentage of blast transformation in the three groups studied. The poor response of the lymphocytes from patients with Downs syndrome to a mitogenic stimulus could reflect an impairment of cellular immune functions in these patients which may be one of the factors contributing to the vulnerability of these patients to repeated or persistent infections.

On the fidelity of transcription by Escherichia coli ribonucleic acid polymerase

The accuracy of transcription has been studied using RNA polymerase from Escherichia coli and synthetic polydeoxynucleotide templates. With the holoenzyme, the highest ratios of non-complementary to complementary bases incorporated into RNA product were observed to be: CTP (1 to 2400), ATP (1 to 9000), UTP (1 to 20,000), GTP (1 to 42,000). The frequency of non-complementary base incorporation into RNA products was not significantly affected by the nature of the template but was found to be primarily determined by the type of non-complementary ribonucleotide. In parallel experiments it was observed that the fidelity of core enzyme was similar to that of holoenzyme. Although removal of sigma factor from holoenzyme had no appreciable effect on non-complementary base-insertion by RNA polymerase, it was observed that the addition of sigma factor to core enzyme stimulated both complementary and non-complementary bases to an equal extent. With poly[d(A-T)]·poly[d(A-T)] as template, nearest-neighbor analysis revealed that CTP was invariably incorporated into the RNA product in juxtaposition with dAMP on the template. Thus, the most frequent error catalyzed by RNA polymerase may be classified as a transition, dA-rU to dA-rC.

[7] Sea urchin nuclear DNA polymerasel☆

Publisher Summary Sea urchin embryos are particularly well suited as a source of DNA polymerase. In the initial cell divisions after fertilization, the rate of DNA synthesis is about 60 times greater than in most other animal cells. This high DNA synthetic activity in the embryos is accompanied by a correspondingly high level of DNA polymerase activity. The biology of this enzyme during early development of the embryo has been studied. It is synthesized during oogenesis and present in the unfertilized egg cytoplasm. With each cell division after fertilization, more and more is sequestered in the nuclei until, by the blastula stage, the majority of the embryos polymerase activity can be isolated with the nuclei. For this reason, the enzyme purification is started with the nuclear fraction. This chapter describes the enzyme activity in the nuclear homogenate and discusses the purification procedure. It is slightly modified to simplify some of the steps, based on the accumulated experience.

Differential heat sensitivity of mammalian DNA polymerases.

Abstract DNA polymerase-β from a variety of mammalian cells is more rapidly inactivated in vitro by elevated temperature than DNA polymerase-α. This differential thermolability is observed between 40 and 50°C and also occurs in the presence of added DNA. It is suggested that heat sensitivity provides a new criterion for delineating DNA polymerase-β and a possible means of examining the function of this enzyme in vivo .

RNA-dependent DNA polymerase: Presence in normal human cells☆

Abstract RNA-dependent DNA polymerase has been reported in oncogenic RNA viruses, as well as in human leukemia cells, suggesting a close relationship between this activity and malignancy. However, we have detected an RNA-dependent DNA polymerase activity in normal human lymphocytes stimulated with phytohemagglutinin, indicating either that this enzyme is not unique to RNA-viruses, or that a viral genome is present in non-malignant human cells. Therefore, the use of this activity as an indication of malignancy or a target for the control of cancer should be approached with caution.

DNA polymerase correlation with DNA replication during transformation of human lymphocytes

Abstract The relationship between DNA polymerase activity and DNA replication during lymphocyte transformation has been studied. On the same sample of cells, both thymidine incorporation and polymerase activity were determined. The induction of DNA polymerase precedes the initiation of DNA replication; thereafter, both activities increase proportionately over a 30-fold range. The deoxynucleotide composition of DNA synthesized in vitro by homogenates of stimulated lymphocytes is similar to that of the added DNA template. The accuracy of dAT copolymer replication is such that equal amounts of dATP and dTTP are incorporated, while if any dCTP or dGTP is incorporated it occurs at a frequency of less than 1 in 103.

Manganese as a mutagenic agent during in vitro DNA synthesis.

Abstract The effect of Mn2+, a known mutagen, on the fidelity of DNA synthesis in vitro by avian myeloblastosis DNA polymerase has been determined. Substitution of Mn2+ for Mg2+ leads to an enhanced incorporation of noncomplementary deoxynucleotides as well as complementary ribonucleotides with either poly (A) or poly (C) as templates. Since this polymerase lacks any detectable deoxyribonuclease activity, the in vitro mutagenic effect of Mn2+ in promoting errors in base-pairing does not result from any diminished proof-reading function.