Initiation of genetic exchanges in λ phage-prophage crosses

Abstract

When Escherichia coli K-12 (.lambda.) lysogens were infected with .lambda. phages, genetic exchanges between phage and prophage occurred at low frequencies (< 0.1% between the markers P3 and P80), but at frequencies > 1% if the infecting phages were first treated with the photosensitizing agent 4,5'',8-trimethylpsoralen and 360 nm light. Exchanges were induced by psoralen damage at about the same frequency in wild-type lysogens and in those carrying recB-, recC-, recF- or lexA-, but at an intermediate frequency in a quadruple mutant carrying recB-, recC-, recF-, sbcB-. Few if any exchanges were induced in lysogens carrying uvrA-, uvrB- or recA-. The increase in the frequency of recombination was presumably due to the psoralen damage in the phage DNA molecules and the action of host cell repair and recombination enzymes. The production of crosslinks in the phage DNA by psoralen and 360 nm light was measured by sedimentation in alkali. It showed 2nd-order kinetics indicative of a 2-photon reaction. First-order kinetics were reported for monoadduct formation. Second-order kinetics, similar to those for crosslink production, were found for genetic exchanges in homoimmune crosses. Presumably, crosslinks, rather than monoadducts, cause most of the exchanges. Because the uvrA+ gene product (UV-endonuclease) was required, recombination was probably initiated by DNA molecules cut at crosslinks. This system was used to show that after the crosslinked phage duplex was cut, .gtoreq. 1 of the subsequent steps.sbd.homologous pairing, cutting and joining.sbd.require the recA+ gene product.