Irving R. Johnston

DNA polymerases of eukaryotes

Although the currently received view of the enzymology of DNA synthesis in prokaryotes is based on a substantial body of information and controversy, it is usually overlooked that serious interest in the DNA polymerases (EC 2.7.7.7.) of both prokaryotes and eukaryotes commenced in the same period, 1956-8. In the following review, a brief summary is given of the situation in prokaryotes before consideration of that in eukaryotes.

Distinct cytoplasmic and nuclear DNA polymerases from rat liver

Interest in enzymes responsible for the synthesis of DNA has accelerated recently following the separation of DNA polymerases I and II in bacteria (for example [I] _ Using aqueous isolation procedures the bulk of the DNA polymerase activity in mammalian systems is found in the cytoplasm [2]. It has also been found that residual activity, not easily removed by washing at low ionic strength, is present in the nucleus [3]. Our interest in pursuing a study of the nuclear and soluble (cytoplasmic) enzymes stemmed from previous work on the zonal fractionation of rat liver nuclei in which we found that nuclear DNA polymerase activity occurred predominantly in nuclei not involved in in vivo DNA synthesis [4]. In this paper we present evidence to show that the major soluble and the nuclear DNA polymerases of rat liver are, in fact, two distinct entities. The minor soluble polymerase appears similar in properties to the nuclear enzyme.

Late replication of the DNA associated with the nuclear membrane

There is considerable light and electron microscopic evidence for the attachment of chromosomal DNA to the nuclear membrane [ 1,2]. Furthermore, several of the reported nuclear membrane prepararations contain DNA [3-61. It is possible, therefore, that a DNA replicating complex, involving the nuclear membrane, may exist in eukaryotes, analogous to the involvement of the cell membrane in bacterial DNA synthesis. Experiments on nuclei from regenerating rat liver, claiming to support this idea, have been published recently [6] . Similar suggestions have also been made from labelling studies on putative DNAmembrane complexes [7,8]. In the work described here, we have isolated nuclei and nuclear membranes, following pulse labelling with 3H-thymidine, from regenerating rat liver at.various times during the first (synchronous) S phase after partial hepatectomy. The results show the nuclear membrane DNA to be predominantly replicated in the latter half of S phase. The evidence argues against the nuclear membrane being the site of all nuclear DNA synthesis.

The role of rat liver nuclear DNA polymerase and its distribution in various classes of liver nuclei

Rate zonal centrifugation has proved a useful technique for the separation of rat liver nuclei into various classes [l] . Studies of labelling in vivo of nuclei following injection of 3H-thymidine and 14C-orotic acid enable an identification of nuclei active in synthesis of DNA and RNA [2] . The distribution of a number of nuclear enzymes including RNA polymerase, NMN-adenylyl-transferase and polyADPR ligase have been investigated [2,3] and certain significant correlations have come to light. One of the most important functions of the nucleus is the replication of its DNA, and DNA polymerase may be involved in this process. If nuclei are isolated from aqueous media only about 20% of the total DNA polymerase is recovered in the nuclear fraction, the greater part being in the cytosol [4] , whereas a substantially greater proportion of the enzyme is found in nuclei prepared in non-aqueous media [ 5,6] . In view of the continuing interest in mammalian DNA polymerase [7,8,9] we have investigated its distribution in fractionated rat liver nuclei and especially its relationship to the nuclei active in vivo in DNA synthesis. These experiments show that the specific activity of DNA polymerase is substantially reduced in nuclei with the maximal labelling.

Molecular asymmetry of rat liver cytoplasmic DNA polymerase

Recent work in several laboratories has shown that the high molecular weight DNA polymerase (polymerase Sl of [ 11) found in high speed supernatants of mammalian cell or tissue homogenates is distinct and separable from the DNA polymerase activity of nuclei [l-S]. Circumstantial evidence implicates this enzyme in the process of replication; for instance its level of activity is very much higher in foetal [6] and regenerating rat [7] livers in both of which the number of cells involved in replication is about twenty times higher that in the adult. Further, in tumours of different growth rates levels of this polymerase, detected by its preference for denatured DNA, correlate well with rates of in vz’vo DNA synthesis determined using [ 3 H]thymidine [7, 81. Although the cytoplasmic location of this polymerase is something of a paradox, it is possible either that it may have leaked from its nuclear environment during aqueous isolation procedures [9], or, as other evidence suggests, the enzyme is normally found in the cytosol in vivo, moving into the nucleus at the beginning of S-phase; this would appear to be the case in both the sea urchin [lo] and in mammalian cells [ 1 l-131. Although substantial purification of the corresponding enzyme from calf thymus [ 141 and human KB cells [IS] has been reported, a more explicit view of the replication process requires that some idea of the molecular weight and sub-unit constitution of polymerase Sl be obtained. As a result of studies undertaken to purify the enzyme from rat liver we wish to report that it exhibits “non-standard” behaviour [ 161 on gel filtration. An estimate of molecular weight is obtained assuming it to be a pure protein and the implications of its deviation from standard behaviour are discussed.

SCM4, a gene that suppresses mutant cdc4 function in budding yeast.

SummaryThe gene SCM4 encodes a protein which suppresses a temperature-sensitive allele of the cell division cycle gene CDC4 in Saccharomyces cerevisiae. SCM4 was cloned on a 1.8 kb BamHI fragment of yeast genomic DNA in the high copy-number vector pJDB207, which results in a 50- to 100-fold increase in the level of the 700 nucleotide SCM4 transcript in vivo. The SCM4 gene encodes a 20.2 kDa protein of 187 aminoacids with a clear tripartite domain structure in which a region rich in charged residues separates two domains of largely uncharged amino acids. Although the apparent allele specificity of cdc4 suppression suggests that the CDC4 and SCM4 proteins interact, disruption of SCM4 demonstrates that the gene product is not essential for mitosis or meiosis; however, it may be a member of a family of related, functionally redundant proteins.

The inhibition of DNA-dependent RNA polymerase of E. coli by Showdomycin

In the course of a search for inhibitors of RNA polymerase (EC 2.7.7.6) from E. coli, it was found that several sulphydryl reagents were able to inactivate the enzyme. Since N-ethylmaleimide was among the compounds found to be inhibitory, it was decided to examine the effectiveness of the naturally occurring maleimide antibiotic, showdomycin. This is an antibiotic produced by Streptomyces showdoensis which inhibits the growth of several bacteria [I] . It has the structure 3+-D-ribofuranosylmaleimide [2] (fig. 1).

Differential N‐ethylmaleimide inhibition of two enzymes of the DNA α‐polymerase‐fraction from calf thymus

The DNA a-polymerase (DNA-nucleotidyltransferase: EC 2.7.7.7.) fraction of calf thymus has been shown to be heterogeneous [l-3] . Using DEAEcellulose chromatography, most preparations can be resolved into three enzymes A,, AZ (both sedimenting at 8.0-8.4 S) and C (7.3 S), detected by means of an activated DNA template. In some preparations a further enzyme, B (5.2 S), is found and probably arises proteolytically from enzyme C [2,4]. A fourth species of enzyme, D, has also been detected by its response to the synthetic template poly(dA) . (dT)i,. Evidence has been presented that enzyme C (mol. wt 1 SO170 X 1 03) can be derived in vitro from the A enzymes (mol. wt 200-230 X 103) by exposure to 2.4 M urea. Whether this treatment eliminates a true sub-unit or a pre-existing fragment created by proteolytic cleavage of the A enzymes either during preparation or in vitro is still not clear [.5] . The additional components is evidently fairly basic in character [6]. The sensitivity of DNA a-polymerase to sulphydryl reagents is commonly used as a criterion to distinguish it from the much less sensitive P-polymerase [7-91. In this paper, however, it is shown that considerable differences exist in the reactivities of cr-polymerase enzymes A and C to inhibition by N-ethylmaleimide (NEM) and to the protective effects of the various substrates.

The effects of some auxins on the level of phosphate esters in Avena sativa coleoptile sections

Abstract 1. 1. The investigation was initiated to study some of the changes in phosphate esters produced by auxins. 2. 2. Using 32 P i it was observed that after 2 h incubation of Avena sativa coleoptile sections in growth-promoting concentrations of 3-indolylacetic acid the ATP/ADP ratio was depressed; this response was considerably diminished after 4–6 h incubation in the auxin. The depression of the ATP/ADP ratio was reduced if the coleoptile sections were preincubated in sucrose. Closer analyses of the time course indicated that the depression of the ATP/ADP ration was detectable 5 min after adding3-indolylacetic acid. 3. 3. 1-Naphthylacetic acid and 2,4-dicholorophenoxyacetic acid apprar to intiate similar falls in the ATP/ADP ratio. 4. 4. 3-Indolylacetic acid increases the labelling of UDPG and decreases that of the hexose phosphates. 1-Naphthylacetic acid invoked a contrary response in the levels of these two compounds while 2,4-dichlorophexyacetic acid had no detectable effect on either of these two compounds. 5. 5. Short-term experiments indicated that the changes in labelling of UDPG and hexose phosphates are detectable 10 min after adding 3-indolylacetic acid or 1-naphthylacetic acid, continue for several hours and then approach controll values. 6. 6. It is shown that growth-promoting concentrations of 3-indolylacetic acid, 1-napthylacetic acid and 2,4-dichlorophenoxyacetic acid increase the rate of incorporation of both 32 P i and [6- 14 C]orotic acid into RNA of Avena sativa coleoptile sections, the time course of this response being similar to that of the above-mentioned changes.

Evidence that the DNA- and poly(A)-dependent DNA polymerase activities of rat liver nuclei are functions of the same enzyme. Some observations on its mode of action

Abstract The low molecular weight DNA polymerase [deoxynucleosidetriphosphate:DNA nucleotidyl transferase (EC 2.7.7.7.)] of rat liver nuclei on purification (1000-fold or more) through three successive steps, retained the ability to utilise both activated DNA and poly(A) · poly(dT) templates. No other species with the ability to copy the polyribonucleotide strand of poly(A) · poly(dT) was observed. Activities on the two types of template decay in parallel on incubation of enzyme at high dilution in the absence of substrate at 37°C. Previous observations on the selective inhibition of the poly(A)-dependent activity by poly(U) do not, however, provide evidence for either a distinct enzyme or for a polyribonucleotide binding site on the enzyme and are shown, in all probability, to be due to formation of a triple helical structure, poly(A) · poly(dT) · poly(U), under the conditions of assay. Finally, the mode of action of the enzyme on decadeoxythymidylic acid initiated-poly(A) and poly(dA) templates is shown at high decadeoxythymidylic acid input levels to be identical. This involves a displacement of poly(dT) strands following incorporation of [ 3 H]dTMP (by gap-filling synthesis) equal to approximately one template equivalent. The rate of synthesis on both synthetic templates at 30°C is remarkably low (less than five nucleotides/min per growing point).