Formation in vitro of infective joint molecules of lambda DNA by T4 gene-32 protein

Abstract

Half molecules of lambda DNA that had been partially digested by lambda exonuclease to expose homologous single strands were rejoined by the action of T4 gene-32 protein at 37 degrees in the presence of Mg(++). Measurements of infectivity in su(-) spheroplasts and sedimentation in sucrose demonstrated the formation of sus(+) joint molecules from two preparations of sheared lambda DNA that carried sus mutations at opposite ends of the genome. The biological activity of joint molecules made by annealing at 75 degrees was diminished by the addition of the gene-32 protein in the absence of Mg(++), and largely restored by the subsequent addition of Mg(++). The specific infectivity of joint molecules made by gene-32 protein at 37 degrees was similar to that of joint molecules made by annealing at 75 degrees . The experimental system described provides a possible model for simulating early steps in genetic recombination.