Isolation of DNA replication complexes from uninfected and adenovirus-infected HeLa cells

• Published in: Journal of molecular biology Vol. 62; no. 1; pp. 65 - 80
• Main Authors: Pearson, George D., Hanawalt, Philip C.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 28.11.1971
• Subjects: Adenoviridae; Bromodeoxyuridine - metabolism; Centrifugation, Density Gradient; Centrifugation, Zonal; DNA Replication; HeLa Cells - metabolism; Humans; Sodium Chloride; Sucrose; Thymidine - metabolism; Tritium; Virus Replication
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/0022-2836(71)90131-8

We have isolated, by zone sedimentation in sucrose shelf gradients, rapidly sedimenting complexes enriched three- to fourfold in newly synthesized DNA from both uninfected and adenovirus-infected HeLa cells. The complexes are observed in the presence of crystalline sodium dodecyl sulfate at pH 8 in 0.5 m-NaCl at 0 °C. Roughly 80% of a three-minute pulse of [ 3H]thymidine sediments in the cellular complex. Over 60% of a five-minute pulse of 5-[ 3H]bromodeoxyuridine is associated with the viral complex while less than 5% of the cellular DNA co-sediments with the viral complex during infection. Thus, the viral genomo may sequester cellular DNA replication sites. The kinetics of labeling indicates that the complexes are sites of DNA synthesis. This has been confirmed by showing that pulse-labeled DNA can be chased from the complexes. The cellular complex is stable to ionic detergents, RNase and several proteases, but not to hog pancrease lipase. The complex is not generated by mixing purified DNA's with crystalline sodium dodecyl sulfate. When added during the isolation procedure, purified viral DNA does not co-sediment with the cellular complex. The rate of adenovirus DNA replication in the presence of 5-bromodeoxyuridine is estimated to be about two μm/minute.