Biochemical assay designed to detect formation of recombination intermediates in vitro

Abstract

A biochemical assay that is designed to detect recombination intermediates formed in vitro is described. The assay measures the fusion of two essentially homologous plasmids, one of which is radioactively labeled and the other of which carries several copies of the lac operator. The fusion product is radioactive and can be bound to a nitrocellulose filter by lac repressor. This assay for genome fusion is rapid and readily applicable to the many fractions that result during enzyme purification. The fused product is not destroyed in the assay and may be recovered from the filter for further analysis by electron microscopy. The product is then seen to consist of figure 8 structures that can be cleaved by the restriction enzyme EcoRI to give chi forms, structures similar to those recovered from recombination-proficient cells. It is expected that this assay will be useful in the purification of the "recombinase-type" activity detected in crude cell lysates. To demonstrate this point, the assay was applied to the protein fractions recovered from a molecular sieve column. The results indicate that the fusion activity has an apparent molecular weight of 50,000--100,000.