A NOVEL ASSAY FOR ILLEGITIMATE RECOMBINATION IN ESCHERICHIA-COLI - STIMULATION OF LAMBDA-BIO TRANSDUCING PHAGE FORMATION BY ULTRAVIOLET-LIGHT AND ITS INDEPENDENCE FROM RECA FUNCTION

Illegitimate recombination takes place between nonhomologous sequences or short homologous sequences at two different sites of DNA(s) and can result in a deletion, duplication, insertion, or translocation of a chromosome. The mechanism of illegitimate recombination is not understood yet, because an appropriate genetic system for the quantitative analysis of illegitimate recombination has not been developed in either prokaryotes or eukaryotes. Illegitimate recombination spontaneously takes place as a rare event in prokaryote as well as in eukaryote, while it is greatly enhanced by treatment with various kinds of DNA-damaging agents. Irradiation of Drosophila with X-rays induces chromosomal aberrations (I). Treatment of bacteriophage T4 with nitrous acid induces deletion mutations in the rII gene (2). Mutations in the DNA polymerase-I structural gene (polA), the exonuclease-I gene (xonA or sbcB), the topoisomerase-III structural gene (mutR or topB), and the histone-like protein-Hla gene (bglY or osmZ) increase the frequency of deletions in the Escherichia coli chromosome (3-6). These observations suggest that illegitimate recombination is induced by DNA-damaging agents and is controlled by cellular genes. We have previously shown that E. coli DNA gyrase, phage T4 topoisomerase, and calf thymus DNA topoisomerase II mediate illegitimate recombination in in vitro systems (7-10). These enzymes are type II topoisomerases that participate in topological changes in circular DNA such as the introduction or removal of supercoils, the formation of catenanes, and the knotting or unknotting of circular DNA (21, 12). The abilities of the enzymes to bind to double-stranded DNA and to make a double-stranded break might have an important role in illegitimate recombination. In fact, we found common sites for recombination and cleavage mediated by DNA gyrase or phage T4 DNA topoisomerase (13, 14). We have also shown that DNA gyrase and T4 DNA topoisomerase participate in deletion formation in vivo (15-17). We have further studied the nature of illegitimate recombination by examining the mechanism of formation of specialized transducing particles of phage lambda. When lysogenic bacteria are induced by ultraviolet (UV) light or heat, almost all of the prophages are excised precisely at both ends, which results in the formation of normal phage particles. On rare occasions, the excision of the prophages does not occur precisely at the ends and E. coli genes adjacent to the prophage DNA (gal or bio) are recombined with the phage genome. The resulting transducing phage DNAs usually comprise the bacterial genes, gal or bio, in addition to the phage genome. We determined nucleotide sequences of the recombination junctions of previously isolated lambda bio phages, indicating that the recombination takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology of 5 to 14 bp (18). The result indicated that the abnormal excision which occurred in the course of formation of the specialized transducing phage is a type of illegitimate recombination. Since the illegitimate recombination during prophage induction takes place between short homologous sequences, it is interesting to ask whether the RecA function is required for the recombination. It has previously been reported that specialized transducing phages are formed independently of RecA protein, which is required for homologous recombination (19, 20). However, these results have not yet proven that specialized transducing phages carrying both cohesive ends arise in the absence of RecA, because Little and Gottesman (21) pointed out that most of the transducing particles in lysates from E. coli recA (it int) lysogen are docL or docR particles with one cohesive end and are therefore formed by cutting the cos site, rather than by illegitimate recombination. In the present paper, we used Spi(-) phenotype (22), instead of bio transduction, for the selection of lambda bio transducing phages. lambda Spi(-) phages are easily distinguishable from the vast majority of normal lambda phage as well as docL or docR particles by plaque assay on an E. coli P2 lysogen. The results showed that treatments with UV light or other DNA-damaging agents enhanced greatly the formation of the Spi(-) phages. RecA function was not required for the illegitimate recombination during the prophage induction. The gyrA or gyrB mutation affected the formation of the Spi(-) phages. Since the present results are consistent with the previous results of illegitimate recombination observed in other systems, our system will provide important clues to understanding of the mechanism of illegitimate recombination.