THE TN7 TRANSPOSASE IS A HETEROMERIC COMPLEX IN WHICH DNA BREAKAGE AND JOINING ACTIVITIES ARE DISTRIBUTED BETWEEN DIFFERENT GENE-PRODUCTS

The bacterial transposon Tn7 translocates by a cut and paste mechanism : excision from the donor site results from double-strand breaks at ea ch end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of t he Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate th at the Tn7 transposase is a heteromeric complex of these proteins, eac h protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA br eakage and joining reactions at the 3' ends of Tn7. Thus the double-st rand breaks that underlie Tn7 excision result from a collaboration bet ween two active sites, one in TnsA and one in TnsB; the same (or a clo sely related) active site in TnsB also mediates the subsequent joining of the 31 ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative D D(35)E motifs blocks catalytic activity. Recombinases of this class re quire a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a T nsA mutant containing a sulfur (cysteine) substitution, we provide evi dence that a divalent metal actually interacts with these acidic amino acids.