Discontinuous DNA replication in mouse P-815 cells.
The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D003851:Deoxyribonucleases; D004586:Electrophoresis; D051379:Mice; D011402:Pronase; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors
D004247:DNA; D003851:Deoxyribonucleases; D011402:Pronase; D013936:Thymidine