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Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

par Rommelaere, Jean ;Faures, Anne ;Errera, Maurice Leo
Référence Journal of Molecular Biology, 90, 3, page (491-508)
Publication Publié, 1974-12

Article révisé par les pairs

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Résumé :

When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since "intermediate DNA" following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease. When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 106) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons. If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, "intermediate DNA" with similar properties is found between the light-heavy and heavy-heavy peaks. © 1974.