Metabolism of Okazaki fragments during simian virus 40 DNA replication.

摘要: Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one three classes. Class I (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and quantitatively identified treating purified with Escherichia coli ligase then measuring fraction joined to chains. Similarly, class II 30%) region single-stranded template (gap) that filled sealed T4 polymerase plus E. ligase, III 50%) RNA primers removed olymerase I, allowing ligase. These results obtained SV40 had been briefly labeled radioactive precursors in either intact cells or isolated nuclei. When nuclei further incubated presence cytosol, converted strands as expected for intermediates synthesis. However, when washed abscence both accumulated despite excision primers: RNA-DNA covalent linkages disappeared at similar rates. data demonstrate existence whole well nuclei, identify unique gap-filling step is not simply an extension chain elongation process concomitant primers. One more factos found addition alpha, are specifically involved ligation steps. The sizes mature (class I) whose synthesis was completed measured gel electrophoresis broadly distributed between 290 nucleotides average length 135 nucleotides. Since 80% 90% Okazaments does occur uniformly spaced intervals along template. During primers, ribonucleotide covalently attached 5' terminus transient intermediates. during adenine 9-beta-D-arabinoside 5'-triphosphate, those blocked III) have originated farthest ends long strands. Thus, appear excised two steps second step, removal final ribonucleotide, being stimulated other used construct comprehensive metabolic pathway initiation, elongation, maturation mammalian replication forks.