Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli

Abstract

Ultraviolet induced pyrimidine dimers remain in the DNA of excision-defective mutants of Escherichia coli for at least several hours after irradiation. If kept in the dark, the survival of colony-forming ability of these mutant bacteria depends upon recombinational repair, a mechanism which is controlled by the gene recA. In u.v.-irradiated bacteria, newly synthesized DNA strands are of low molecular weight, but these strands are subsequently joined into high molecular weight chains, as is shown by sedimenting the bacterial DNA in alkaline sucrose gradients. These longer chains may contribute to cell survival by acting as templates for the synthesis of high molecular weight strands during further replication. At times greater than 90 minutes after irradiation, the molecular weight of newly synthesized DNA covers a wide range and extends fully to the maximum found with unirradiated cells, thus indicating the presence of high molecular weight templates virtually free from damaged bases. Evidently the u.v. photoproducts tend to remain in the original strands where they were formed, rather than becoming distributed equally among all the strands with each subsequent replication. Recombinational repair may depend upon genetic exchanges between sister duplexes. To see whether such exchanges could be detected after u.v.-irradiation, cells were grown for several generations in medium containing 13C and 15N. They were then exposed to various doses of ultraviolet light and grown for less than a generation in light medium containing [ 3H]thymidine, so that part of their DNA was hybrid in density, with the 3H label in the light strand. Under these conditions, light 3H-labeled chains will become linked to heavy strands if exchanges occur between sister duplexes. The DNA was extracted from unirradiated cells, heat denatured and centrifuged in neutral cesium chloride gradients, and was found to separate into heavy and light bands. Denatured DNA from u.v.-irradiated cells did not separate completely, but contained strands of intermediate density which separated into heavy and light components only after shearing to 5 × 10 5 molecular weight, thus indicating that segments of molecular weight greater than 5 × 10 5 had been exchanged. To judge from the fraction of the 3H-labeled molecules that were of intermediate density, one exchange occurred for every one to two pyrimidine dimers in the DNA replicated in the 3H-labeled medium. Sister exchanges involving a single strand of each duplex, may insert the correct bases sequence into the gap opposite each pyrimidine dimer, and thus promote the reconstruction of a genome with the complete base sequence needed for the survival of colony-forming ability.