DNA Polymerases of Rat Liver

Abstract

Three distinct DNA-dependent DNA polymerase activitives have been partially purified from normal rat liver. Soluble activities are separable into two distinct fractions (P1 and P2) by phosphocellulose chromatography. A low-molecular-weight DNA polymerase was isolated from purified nuclei. The enzymes were characterized according to chromatographic and sedimentation behavior, enzymological properties, and response to various inhibitors. The results indicate that fraction P1 corresponds to the high-molecular-weight enzyme and suggest that polymerase P2 may be derived from partial dissociation of the high-molecular-weight enzyme. The molecular weight of polymerase P1 was estimated to be about 250000 by Sephadex column chromatography. Both fraction P2 and nuclear DNA polymerase appeared to be low-molecular-weight enzymes. However, the molecular size of these activities was apparently different. The estimated molecular weights of nuclear and P2 enzyme are about 40000 and 25000, respectively.As with the nuclear enzyme, polymerase P2 (but not P1) appeared to be free of detectable exonuclease activity. All of these polymerases showed a marked preference for initiated polydeoxyribonucleotide templates. The rat liver polymerases differed in their ability to use poly[d(A-T)] primer-template, as is shown by the ratios of their activity with this synthetic polymer to that with activated DNA: 0.5, 2.75, and 1.34 for P1, P2, and nuclear polymerase, respectively. Denatured DNA was a poor template for both enzymes P1 and P2, but it was inert as template for the nuclear enzyme.Although each of these polymerases required all four deoxynucleoside triphosphates for maximal activity, they catalyzed a high rate of synthesis in the absence of one or more deoxynucleoside triphosphates. Such a 'limited' synthesis was much more extensive for polymerase P2 and nuclear enzyme than for P1. Polymerase P1 was the most sensitive of the three to sulphydryl reagents, ethidium bromide, heparin, and single-stranded DNA. The responses of P2 and nuclear enzymes to various inhibitors were very similar. However, these two enzymes respond differently to heat and high ionic strength.The existence of multiple species of DNA polymerase has been demonstrated in a wide variety of eukariotic cells and tissues [1,2]. The extent and nature of multiplicity, the actual cellular localization, and the physiological function of enzyme activities are matters of current investigation. The eukaryotic DNA poly- ~ ~~Abbreviations. dNTPs, deoxyribonucleoside triphosphates; poly[d(A-T)], alternating double-helical deoxyadenylate-thymidylate copolymer; poly(dA) . (dT),,, polydeoxyadenylic acid associated with decathymidylic acid with a base-ratio of 1 : 1 : poly(rA) . (dT),,, polyadenylic acid associated with decathymidylic acid with a baseratio of 1 : 1 ; (dT),,, decathymidylic acid; poly(dA), polydeoxyadenylic acid.

Effect of drugs on deoxyribonucleic acid synthesis in isolated mammalian cell nuclei. Comparison with partially purified deoxyribonucleic acid polymerases

“…The stimulatory effect of low concentrations of ethidium bromide on DNA synthesis by isolated nuclei is presumably a consequence of a change in the conformation of DNA caused by this intercalating dye (Paoletti, 1978). A similar stimulation of DNA and RNA synthesis in vitro has been reported (Meyer et al, 1972; R. R. Meyer, personal communication), and ethidium bromide in vivo at 0.1-0.5,ug/ml was shown to stimulate BHK-cell nuclear DNA synthesis by 250% (Zunino et al, 1975). We did not detect a clear stimulation of DNA synthesis by using the partially purified polymerases and 'activated' DNA as template, nor was there a difference in the response of polymerase-a and -fi to ethidium bromide, unlike a previous report showing the a-enzyme to be more sensitive to this drug than the fl-enzyme (Zunino et al, 1975).…”

The Interaction of Daunomycin with Polydeoxynucleotides

“…IHowever, the observation that the poly(dA) . poly(dT) preparation used in this work was inert as template for rat liver DNA polymerase CI (which showed a marked preference for initiated polydeoxynucleotide templates [17]) suggests that the above possibility is remote.…”

“…Enzyme Assays-E. coli D N A polymerase I The enzyme used was a highly purified preparation as specified by Jovin et al (161. The standard assay contained, in a final volume of 0.25 ml: 60 mM Tris-HCI (pH 7.4), 6 mM MgC12, 10 mM 2-mercaptoethanol, 100 pM each of complementary deoxyribonucleoside triphosphates (including a 3H-labelled dNTP, specific activity 40 counts min-' pmol-'), 80 pM primer-template, and 0.015 unit of DNA polymerase. After 10min of incubation at 37 "C, the acid-precipitable material was measured as previously described [17]. One unit of DNA polymerase is defined as the amount catalysing the incorporation of 10 nmol of dTMP into acid-insoluble material during 10min of incubation at 37°C under our standard assay conditions with 'activated' calf thymus DNA [18] as primer-template.…”

“…After 10min of incubation at 37 "C, the acid-precipitable material was measured as previously described [17]. One unit of DNA polymerase is defined as the amount catalysing the incorporation of 10 nmol of dTMP into acid-insoluble material during 10min of incubation at 37°C under our standard assay conditions with 'activated' calf thymus DNA [18] as primer-template.…”