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Genetics, Vol 129, 1007-1020, Copyright © 1991
INVESTIGATIONS |
Recombinagenic Processing of UV-Light Photoproducts in Nonreplicating Phage DNA by the Escherichia coli Methyl-Directed Mismatch Repair System
W. Y. Feng, E. Lee and J. B. Hays
Department of Agricultural Chemistry, Oregon State University, Oregon State University, Corvallis, Oregon 97331
Nonreplicating {lambda} phage DNA in homoimmune Escherichia coli lysogens provides a useful model system for study of processes that activate DNA for homologous recombination. We measured recombination by extracting phage DNA from infected cells, using it to transfect recA recipient cells, and scoring the frequency of recombinant infective centers. With unirradiated phage, recombinant frequencies were less than 0.1%. However, recombination could be increased over 300-fold by prior UV irradiation of the phages. The dependence of recombination on UvrA function varied greatly with UV dose. With phage irradiated to 20 J/m(2), recombinant frequencies in repressed infections of uvr(+) bacteria were one-fifth those in uvrA infections; with phages irradiated to 100 J/m(2), frequencies in uvr(+) infections were thirty times higher than in uvrA infections. Most UV-stimulated recombination in uvrA infections appeared to depend on the bacterial methyl-directed mismatch-repair system: frequencies were depressed 5-20-fold in uvrA bacteria also lacking MutH, MutL or MutS functions, and recombinant frequencies decreased with increasing GATC-adenine methylation of phage stocks. The biological activity of nonreplicating UV-irradiated phage DNA declined with time after infection of uvrA cells; this decline was photoproduct-dependent, more marked for undermethylated than overmethylated phage DNA, and depended on host MutHLS functions. In uvr(+) bacteria, where the UvrABC system provided an alternative, apparently less efficient, route to recombinagenic DNA, UV-stimulated recombinant frequencies were about twice as high in mutH or mutLS as in mut(+) cells, in agreement with hyper-rec mut effects previously described by others.
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