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Nucleic Acids Research, 1977, Vol. 4, No. 6 2083-2098
© 1977

Articles

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

Friedrich Wanka, Rob M.G.M.E. Brouns, Jan M.A. Aelen and Jelle Eygensteyn

Department of Chemical Cytology, University of Nijmegen Nijmegen, The Netherlands

Received April 14, 1977. 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.


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