No abstract

Bacterial DNA replication involves as intermediates structures with single-stranded regions at the growing point (1-3). Newly synthesized, pulse-labeled DNA that behaves on hydroxyapatite and/or nitrocellulose as though it contained single-stranded regions has also been observed in mammalian cells (4-8). Chromatography on benzoylated, naphthoylated DEAE-cellulose permits the separation of such single-strandcontaining intermediates in DNA synthesis that accumulate after treatment with the monofunctional alkylating agent, methyl methanesulfonate (MMS, ref. 9). We report here the isolation from HEp. 2 cells incubated with BrdU of a fraction of newly synthesized DNA whose banding characteristics in CsCl gradients and response to incubation with a singlestrand-specific nuclease indicate that it contains singlestranded regions in both parental and newly synthesized DNA. Treatment of cells with MMS at concentrations that inhibit DNA synthesis increases the proportion of pulselabeled material appearing in this fraction. We propose that the fraction collected at a density of 1.715 g/cm8 in CsCl is enriched for normal intermediates in DNA synthesis and that alkylation-induced lesions have the effect of immobilizing and accumulating a replicating structure. into BrdU-FdU medium to a concentration of 10 mM. Cells were harvested as described (11).DNA Extraction. Cells were harvested and washed in phosphate-buffered saline (11), washed in 0.15 M NaCl + 0.015 M sodium citrate, and resuspended in saline-citrate at approximately 4.0 X 106/ml. Single-stranded, newly synthesized DNA is preferentially adsorbed to the interface of organic solvent extractions (13,14). Our preparations must, therefore, be done at high cell density (15), which makes it possible to avoid the losses in recovery reported by Hanawalt and Ray (16). Sodium dodecyl sulfate was added to 0.2%, and extraction-was carried out as reported (11).CsCl Density Centrifugation. DNA (30-60 gg), sheared by passage three times through a 22-gauge needle, was centrifuged though a CsCl gradient that was collected and processed as described (11). Alkaline gradients were prepared by the dropwise addition of 0.1 M NaOH to the DNA sample to bring the pH to 12 before addition of CsCl.Exonuclease Treatment. Fractions from CsCl gradients were pooled and dialyzed against 0.13 M glycine buffer, pH 8.4. Samples were incubated in a reaction mixture of 1.5 ml with 3 units/ml of Escherichia coli exonuclease I (supplied by Dr. N. Cozzarelli) in 13 mM MgCl2 and 3.3 mM 2-mercaptoethanol for 1.5 hr at 37°. Nuclease susceptibility was measured by comparison of the radioactivity solubilized by cold and hot 5% trichloroacetic acid. This preparation of exonuclease I, although completely specific for single-stranded DNA, includes some endonuclease activity (9). At least 90% of denatured DNA was converted to acid-soluble fragments after treatment with enzyme. 1969Abbreviation: MMS, methyl methanesulfonate.

No abstract