Detection by density equilibrium centrifugation of recombinant-like DNA molecules in somatic mammalian cells.

Abstract

When the nuclear DNA of Chinese hamster cells grown in the presence of bromo-deoxyuridine for one or less generation is analysed in an alkaline CsCl density gradient, a small fraction of replicated DNA can be found at densities intermediate between those of the light and heavy strands. The labelling pattern of these displaced molecules and their segregation into fully light and heavy fragments after sonication indicate that they are formed by the covalent joining of light and heavy segments. The duplexes giving rise to displaced DNA upon denaturation are mostly found at the position of light-heavy DNA in a neutral gradient; a minor component, however, bands at densities between light-heavy and heavy-heavy DNA. The high degree of renaturability of the former molecules suggests that they contain cross-links between the complementary strands; the latter, on the contrary, are likely to comprise a segment substituted in both strands, which is interpreted as being a heteroduplex resulting from somatic crossing-over between uninemic sister chromatids. Although the frequency of these recombinant-like molecules is increased several-fold after u.v. irradiation, the data do not support any direct implication of these exchanges in a postreplication repair mechanism of the type found in bacteria.