Discontinuous DNA Replication in Mouse P‐815 Cells

Abstract

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 ...

Discontinuous DNA Synthesis in Mammalian Cells

“…, 1973;Goldstein & Rutman, 1973;Berger & Huang, 1974;Friedman et a1. , 1975;Gautschi & Clarkson, 1975;Mendelsohn et a1., 1975;Tseng & Goulian, 1975a) and in vitro (Kidwell & Mueller, 1969;Fox et al, 1973;Friedman, 1974;Tseng & Goulian, 1975a).…”

“…, 1971;Fareed & Salzman & Thoren, 1973;Francke & Hunter, 1974a, b;Kriegstein & Hogness, 1974;Pigiet et a1., 1974;Qasba, 1974b;Friedman et a1., 1975;Gautschi & Clarkson, 1975;Mendelsohn 8t a1., 1975;Tseng & Goulian, 1975a) to 10-20 nucleotides (Schandl & Taylor, 1969Taylor et a1., 1970;Schandl, 1972). I n this thesis I present evidence that the Okazaki fragments of mammalian cells are about 100 nucleotides long, and I discuss why some researchers found other values.…”

“…Whether the other strand is also made discontinuously is still very much an open question. On the basis of the assumption that more than 50% of acid-precipitable radioactivity in small pieces after a short pulse-label indicates that both strands are being synthesized discontinuously, many groups have claimed to observe totally discontinuous synthesis (Okazaki et al , 1968a(Okazaki et al , , h, 1973Sadowski et al, 1968;Yudelevich et al, 1968;Fareed et al, 1973;Goldstein & Rutman, 1973;Gottesman et al, 1973a;Hess et al, 1973;Kohn et al J 1974;Gautschi & Clarkson, 1975;Mendelsohn et a1. , 1975;Tseng & Goulian, 1975a).…”

“…Some report that they are mainly si ngle stranded after lysis (Okazaki et al, 19 68a;Painter & Schaefer, 1969;Sato et a1., 1970;Cheevers et a1., 1972;Mendelsohn et a1., 1975) while others say they are not (Tsukada et a1. , 1968;Yudel evich et a1., 1968;Fareed & Salzman, 1972;Gautschi & Clarkson, 1975 (Sugino et a1., 1972;Hirose et al, 1973;Okazaki et a1., 1973Okazaki et a1., , 1975, colicinogenic Factor El (Blair et a1., 1972), phage (Brutlag et a1., 1971;Miller, 1972;Schekman et a1., 1972;Wickner et a1., 1972), Physarum (Waqar & Huberman, 1973, 1975a, polyoma (Magnusson et a1., 1973;Sadoff & Cheevers, 1973;Hunter & Francke, 1974b;Pigiet et a1., 1974;Reichard et a1., 1974), SV40 (Qasba, 1974b), 24 and mammalian cells (Sato et al, 1972;Fox et al, 1973;Taylor et al , 1973b;Neubart & Bases, 1974;Tseng & Gaulian, 1975b;Qaqar & Huberman, 1975b). Evidence for RNA has been found either by CSzSO4 equilibrium density gradient centrifugation or by nearest neighbor analysis.…”

“…A few groups do not find RNA, but speculate that their pulse times may have been too long to observe attached RNA or that RNA may have been removed during treatment of the DNA samples (Berger and Huang, 1974;Gautschi and Clarkson, 1975).…”