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 (less than 0.1% between the markers P3 and P80), but at frequencies above 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 second-order kinetics indicative of a two-photon reaction. In contrast, first-order kinetics had been 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, it is likely that recombination was initiated by DNA molecules cut at crosslinks. This system has been used to show that after the crosslinked phage duplex has been cut, one or more of the subsequent steps-homologous pairing, cutting, and joining-require the recA(+) gene product.