Production of DNA bifilarly substituted with bromodeoxyuridine in the first round of synthesis: branch migration during isolation of cellular DNA.
Incubation of human lymphoid cells with bromodeoxyuridine (BrdUrd) for short periods produces three classes of DNA containing analog: DNAHL (hybrid DNA, density approximately equal to 1.75 g/cm3), DNAint (intermediate density DNA, density approximately equal to 1.71 g/cm3), and DNAHH (DNA with both strands containing analog, density approximately equal to 1.80 g/cm3). Preparations of DNAint yield DNAHH after extensive shearing and/or treatment with single strand specific endonuclease. Cross-linking of pulse-labeled (BrdUrd + 3HdT) DNA in cells by treatment with trioxsalen and near UV light before lysis prevents the appearance of DNAHH.Cross-linking after lysis has little effect. A large fraction of DNAHH 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 DNAHH obtained from caffeine-treated cells. We conclude that most DNAHH arises from in vitro branch migration in isolated DNA growing points.
D001973:Bromodeoxyuridine / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D005467:Floxuridine / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D010777:Photochemistry; D014307:Trioxsalen
D005467:Floxuridine; D002110:Caffeine; D004247:DNA; D001973:Bromodeoxyuridine; D014307:Trioxsalen