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.[3H]-Thymidine pulses of 2-8 min at 37 "C predominantly label molecules of 20-60 S. With 30-s pulses at 2 5 T , all the [3H]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, 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 Cs2S04 gradients or after incubation with polynucleotide kinase and [Y-~~PIATP.Semiconservative replication of Escherichia coli DNA and T4 phage DNA is discontinuous and involves the 5'-3' synthesis of 9-S precursor molecules, Okazaki fragments, on both daughter strands at each replication fork [l -31. Similar replication intermediates of various sizes have been described in other bacteria and phages as well as in eukaryotes (see [4]). These intermediates also occur during replication of Simian virus 40 (SV40) [5] and polyoma virus DNA [6,7] ; they are 3 -5 S long.Recently, "primer" RNA molecules covalently attached to nascent Okazaki fragments have been demonstrated in E. coli [3] and in isolated nuclei synthesizing polyoma DNA [6,8]. Evidence for similar RNA-DNA copolymers was also found in Physarum polycephafum [9,10] and in an established cell line of human lymphocytes [ll].The reports on replication intermediates in mammalian cells [lo, 12-20], however, vary with respect to estimations of size, lifetime, and buoyant density. Structure also became a point of interest when, as in previous reports [15,19,21], newly synthesized DNA was recovered from lysates as single-stranded molecules without using specific conditions for DNA denaturation,In order to define different characteristics of DNA replication in the same system, we studied mouse Their fiber autoradiographic studies suggest that nuclear DNA of mammalian cells is composed of tandemly joined sections (15 to 60 pm) which are replicated bidirectionally from one origin [24]. The two growing points in each replication unit form forklike structures which are displaced from the origin at an estimated rate of about 1 pm/min. Units adjacent to each other replicate predominantly synchronously. These replication units will be referred to in this paper as "replicons". (Note that this definition of the term "replicon" is not identical to the original one for bacterial systems [25]. One should emphasize that in mammalian cells the timing of operation of individual "replicons" is programmed [26].) In HeLa cells we found replication intermediates [22] with sizes corresponding on the average to about half the length of autoradiographically visualized ...
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