There exists a distinct stage during mammalian DNA synthesis immediately after joining of replication intermediates.
We describe an approach, using alkaline cell lysis and digestion with nuclease S1, which permits to distinguish between newly ligated DNA and the DNA of mature chromatin. When cells with steady-state labelled DNA (mature DNA) are analyzed, the results show labelled "nucleosomal-sized" DNA. However, when DNA of cells pulse-labelled with thymidine for 45 seconds is examined one can detect only large DNA. The newly ligated DNA is not reduced to "nucleosomal-sized" DNA by nuclease S1. When the large DNA is denatured in formamide one can detect 10 kb DNA fragments. Furthermore in pulse-chase experiments there appear, after formamide-treatment, increasing amounts of "nucleosomal-sized" DNA with a parallel decrease in the amount of 10 kb DNA fragments. Hence the newly ligated, large, DNA differs from mature DNA and represents a distinct stage during DNA replication.
D002478:Cells, Cultured; D002843:Chromatin / Q000648:ultrastructure; D004261:DNA Replication; D004720:Endonucleases; D005559:Formamides; D006801:Humans; D008545:Melanoma; D008836:Micrococcal Nuclease; D009707:Nucleosomes / Q000648:ultrastructure*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014965:X-Rays
D002843:Chromatin; D005559:Formamides; D009707:Nucleosomes; C031066:formamide; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008836:Micrococcal Nuclease