Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA

Abstract

Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNA _HL (hybrid DNA, density≅1.75 g/cm ³ ), DNA _int (intermediate density DNA, density≅1.71 g/cm ³ ), and DNA _HH (DNA with both strands containing analog, density≅1.80 g/cm ³ ). Preparations of DNA _int yield DNA _HH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (Brdurd+ ³ HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNA _HH . Cross-linking, after lysis has little effect. A large fraction of DNA _HH is obtained after incubation of cells with caffeine. Extraction of DNA at high salt concentration or cross-linking with trioxsalen and near UV light drastically reduced the amount of DNA _HH obtained from caffeine-treated cells. We conclude that most DNA _HH arises from in vitro branch migration in isolated DNA growing points.