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JBC, Vol. 254, Issue 22, 11495-11504, Nov, 1979
S. Anderson and M. L. DePamphilis
Essentially all of the Okazaki fragments on replicating Simian virus 40
(SV40)DNA could be grouped into one of three classes. Class I Okazaki
fragments (about 20%) were separated from longer nascent DNA chains by a
single phosphodiester bond interruption (nick) and were quantitatively
identified by treating purified replicating DNA with Escherichia coli DNA
ligase and then measuring the fraction of Okazaki fragments joined to
longer nascent DNA chains. Similarly, class II Okazaki fragments (about
30%) were separated by a region of single-stranded DNA template (gap) that
could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase,
and class III fragments (about 50%) were separated by RNA primers that
could be removed with E. coli DNA olymerase I, allowing the fragments to be
joined with E. coli DNA ligase. These results were obtained with
replicating SV40 DNA that had been briefly labeled with radioactive
precursors in either intact cells or isolated nuclei. When isolated nuclei
were further incubated in the presence of cytosol, all of the Okazaki
fragments were converted into longer DNA strands as expected for
intermediates in DNA synthesis. However, when washed nuclei were incubated
in the abscence of cytosol, both class I and class II Okazaki fragments
accumulated despite the excision of RNA primers: class III Okazaki
fragments and RNA-DNA covalent linkages both disappeared at similar rates.
These data demonstrate the existence of RNA primers in whole cells as well
as in isolated nuclei, and identify a unique gap-filling step that is not
simply an extension of the DNA chain elongation process concomitant with
the excision of RNA primers. One or more factos found in cytosol, in
addition to DNA polymerase alpha, are specifically involved in the
gap-filling and ligation steps. The sizes of mature Okazaki fragments
(class I) and Okazaki fragments whose synthesis was completed by T4 DNA
polymerase were measured by gel electrophoresis and found to be broadly
distributed between 40 and 290 nucleotides with an average length of 135
nucleotides. Since 80% and 90% of the Okazaments does not occur at
uniformly spaced intervals along the DNA template. During the excision of
RNA primers, nascent DNA chains with a single ribonucleotide covalently
attached to the 5' terminus were identified as transient intermediates.
These intermediates accumulated during excision of RNA primers in the
presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki
fragments blocked by RNA primers (class III) were found to have originated
the farthest from the 5' ends of long nascent DNA strands. Thus, RNA
primers appear to be excised in two steps with the second step, removal of
the final ribonucleotide, being stimulated by concomitant DNA synthesis.
These and other data were used to construct a comprehensive metabolic
pathway for the initiation, elongation, and maturation of Okazaki fragments
at mammalian DNA replication forks.
Metabolism of Okazaki fragments during simian virus 40 DNA replication
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