The origin of nascent single-stranded DNA extracted from mammalian cells.
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 sedimenting fraction of preferentially pulse labelled DNA. The DNA of this zone was further analysed by chromatography on hydroxy-apatite, banding in CsCl 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 microgram/ml pronase. The results suggest that nascent single-strands and nascent-nascent duplexes are released from the forks of replicating DNA by branch migration. Pre-labelled single strands may be released by the same 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.
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002500:Centrifugation, Isopycnic; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000302:isolation & purification*; D008099:Liver / Q000032:analysis*; D013936:Thymidine / Q000378:metabolism
D004277:DNA, Single-Stranded; D013936:Thymidine