Nuclear Membranes from Mammalian Liver, V. On the Question of DNA Polymerase Activities Associated with the Nuclear Envelope

Abstract

Deoxyribonucleoside triphosphate incorporating activity was assayed with isolated rat and pig liver nuclei as well as with nuclear subfractions such as purified nuclear membranes, chromatin reconstituted from nuclear high concentration salt extract, and native eu-and heterochromatin. With the intact isolated nuclei, the incorporation of the precursor into the membraneattached DNA proceeded at a lower rate to give a very early plateau. DNA polymerase activity proved to be totally extractable with high salt concentrations. In contrast to the fractions containing chromatin, isolated nuclear membranes had no incorporating activity. This demonstrates that DNA polymerase is unlikely to be a membrane component bound in a non-ionic form. The nuclear membrane proteins in such assays revealed a high adsorptive affinity for the nucleoside triphosphates used. The results are discussed against the background of the current hypotheses of an involvement of the inner nuclear membrane in replicating events and support the view that the replicase is constitutively attached to the template rather than to the membrane. Zellkermnembranen aus Säugerleber, V: Zur Frage der an die Zellkernhülle assoziierten D N A-PolymeraseAktivitätenZusammenfassung: Der Einbau von Desoxyribonucleosidtriphosphaten (dTTP, dATP) in DNA wurde in einem "DNA-Polymerase-Ansatz" bei folgenden Fraktionen aus Ratten-und Schweineleber geprüft: isolierte Zellkerne, MikrosomenMembranen, gereinigte Kernmembranen, natives (Eu-und Hetero-) und rekonstituiertes Chromatin. Bei den isolierten Kernen werden die Vorstufen schneller und über längere Zeit in nicht-Kernmembran-ständige DNA eingebaut. Während bei allen Chromatin-Fraktionen starker Einbau gemessen wurde, der die Charakteristika für Kern-DNA-Polymerase-Aktivitäten aufwies, konnte bei den Membranfraktionen kein Einbau festgestellt werden. Die untersuchten Membranfraktionen zeigten aber eine hohe (nicht-enzymatische) Bindungsaffinität für die eingesetzten Nucleosidtriphosphate. Die Ergebnisse zeigen, daß DNAPolymerase kein konstitutioneller Bestandteil der Kernhülle ist, sondern aus Kernen mit hoher

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.

“…The matrix-bound alpha polymerase endogenous and exogenous template-driven activities were depleted in the nuclear lamina when expressed as a percent of total matrix activity or on a specific activity basis (Table III). These results agree with earlier reports that suggested the absence of DNA polymerase activity in isolated nuclear envelopes (23,38). In contrast, the small amount of nuclear DNA polymerase beta activity associated with the isolated nuclear matrix (~5 % of total nuclear activity; see also references 55-57) had a strikingly different distribution.…”

Nuclear envelopes: Structure and biochemistry of the nuclear envelope

“…However, these observations have been refuted by other authors (e.g. Deumling & Franke 1972;Kay, Fraser & Johnston 1973). So far only the occurrence of membraneassociated nuclear DNA and, perhaps, of a special nuclear envelope RNA (Franke et al 1973;Franke & Scheer 1974;Kasper 1974) might serve as a chemical component distinguishing the nuclear envelope from other cellular membranes but even here the literature is controversial (compare, for example, Kay, Haines & Johnston 1971;Fakan et al 1972;Mizuno, Stoops & Sinha 1971;Mizuno, Stoops & Pfeiffer 1971 b ;) an especially in view of experiments of Kubinski, Gibbs & Kasper (1972), who demonstrated that some endomembranes are capable of binding free nucleic acids in a firm (and not yet under stood) manner.…”

“…association with isolated nuclear membranes (Kay 1971;Deumling & Franke 1972;Fakan et al 1972;Franke et al 1973 a). The intim ate association of some DNA with the nuclear envelope can also be demonstrated directly in nuclear envelope fragments (figure 31) which have been purified by flotation in 4 m CsCI and in sucrose gradients (for details see Franke etal.…”