Enhanced recombination associated with the presence of insertion and deletion mutations in poxvirus-infected cells

Genetic crosses were performed in cells infected by Shope fibroma virus in order to determine whether poxviral recombination is influenced by the types of mutations participating in a cross. Infected cells were cotransfected with marked bacteriophage lambda DNAs and, after DNA recovery, the substrates were packaged into infectious particles using in vitro packaging extracts. This made it possible to analyze the products of poxviral recombination systems using lambda mutants plated on Escherichia coli. It was observed that, regardless of the kind of substitution, all point mutations recombined in virus-infected cells with frequency dependent only on distance. In contrast, when crosses were performed when one of the mutations was an insertion or a deletion, recombination was stimulated by amounts ranging from 20 to 100%. This phenomenon was not dependent on the size of the nonhomology, was observed across a range of marker separations, and was not due to packaging, plating, or reversion artifacts. The best explanation for this marker effect is that regions of nonhomology act to block the migration of strand-exchange intermediates thereby forcing exchange in the immediate vicinity of a nonhomology. This hypothesis is compatible with the failure to detect loop-heteroduplex intermediates and the specific effects of altered marker configurations in four-factor crosses. It is also compatible with the observation that when two nonhomologies were crossed, they appeared to inhibit recombination in the region located between the two elements. These observations suggest a possible way in which poxviral recombination might be targetted into, or away from, regions of particular interest. The similarities between these observations and data derived from phage and fungal experiments also provide fundamental insights into the mechanism of poxviral recombination.