Studies on the nature of replicating DNA of HeLa cells

Abstract

Replicating DNA from synchronized HeLa cells pulse labeled with radioactive thymidine differed from non-replicating DNA by its partition to the interphase fraction during extraction with phenol or chloroform. Pulse labeling followed by a chase with non-isotopic thymidine demonstrated that DNA of the interphase fraction was converted to a form which was extractable in the aqueous phase. Blocking DNA synthesis with hydroxyurea prevented this conversion. Sonication of the interphase DNA demonstrated that the property causing the extraction of the DNA into the interphase was localized at or near the site of active replication. Centrifugation of the replicating DNA isolated from the phenol-water interphase revealed that the labeled material separated into two fractions: one which sedimented rapidly to the bottom of a sucrose gradient, but floated in CsCl density gradient; and a fraction which sedimented slightly slower than non-replicating DNA in sucrose gradient but exhibited the same buoyant density in CsCl. The rapidly sedimenting material was converted to a slowly sedimenting material with the same buoyant density as non-replicating DNA by heat, alkali, or sonication, but was unaffected by pronase. Evidence for attachment of a cellular component to replicating DNA is discussed.