The origin of nascent single-stranded DNA extracted from mammalian cells

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Abstract
In vitro cultured bovine liver cells were labelled with radioactive thymidine and dissolved in 0.5% sodium dodecyl sulphate. Centrifugation of the lysate through sucrose gradients in a zonal rotor revealed a slowly sedimen-ting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CaCl density gradients, and sedimentation in neutral and alkaline sucrose gradients. It contained, besides small amounts of fragmented bulk DNA., single-stranded nascent DNA and single-stranded pre-labelled DNA which could be separated from each other by using BrdU as a density label. The density labelling also revealed small amounts of nascent-nascent DNA duplexes. The slowly sedimenting fraction was practically absent from cell lysates which were prepared in 2 M NaCl - 50 μg/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating UNA by branch migration. Pre-labelled single strands may be released by the same mechanism, but the in vivo structure from which they originate has yet to be elucidated.