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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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Single and double‐stranded polynucleotides of thymine and cytosine have been used to analyse the photoproducts produced by irradiation with far or near UV light. Reversed‐phase high performance liquid chromatography was used to detect and quantify cyclobutane dimers and Pyr(6–4)Pyo adducts produced by 254 nm. At 320 nm 10‐times less Thy(5–6)Thy dimers and one half the number of Cyt(5–6)Cyt dimers were induced; no Pyr(6–4)Pyo adducts were evident. HPLC has recently been applied to the isolation and characterization of various nucleic acid substituents and their UV‐photoproducts. The relative retention times of pyrimidines and their UV photoproducts on HPLC reflect differences in the hydrophobicity of the compounds being separated. Hence, the more hydrophobic (less polar) a compound is, the greater its capacity to bind to the column and the greater its retention time; for T o T, C<>T and C<>C dimers this difference may result from variations in the number of methyl moieties (Cadet et al., 1983). The retention time of the compound also depends on its stereochemistry. Separation of the four stereoisomers of T<>T by HPLC shows that compounds which are molecular equivalents can be more or less accessible to the non‐polar stationary phase depending on their conformation (Cadet, 1980). The relatively long retention times of the bipyrimidine photoadducts suggest a structural configuration which allows greater access to the hydrophobic moieties of the molecule. It is intriguing to consider that this difference in conformation may also be reflected in DNA–protein interactions such as binding by UV‐endonucleases or antibodies directed against UV photodamage. Proteins can be used as sensitive probes for photoproduct induction and repair but an accurate evaluation of their specificity is required. It was the intent of this paper not only to compare the induction parameters for various dimers in pyrimidine homo‐polymers but to provide controlled substrates which can be used to define the relative binding efficiencies of repair enzymes and antibodies for different types of photoproducts.

Abstract— The removal of cyclobutane dimers and Pyr(6–4)Pyo photoproducts from the DNA of UV‐irradiated ICR 2A frog cells was determined by radioimmunoassay. In the absence of photoreactivat‐ing light, 15% of the cyclobutane dimers and 60% of the (6–4) photoproducts were removed 24 h post‐irradiation with 10 J m−2, Exposure to 30 kJ m−2 photoreactivating light resulted in removal of 80% of the cyclobutane dimers and an enhanced rate of repair of (6–4) photoproducts, resulting in a loss of 50% of these lesions in 3 h. The preferential removal of (6–4) photoproducts by excision repair resembles previously published data for mammalian cells.