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 each end of Tn7 and target insertion results from joining of the exposed 3' Tn7 tips to the target DNA. Through site-directed mutagenesis of the Tn7-encoded transposition proteins TnsA and TnsB, we demonstrate that the Tn7 transposase is a heteromeric complex of these proteins, each protein executing different DNA processing reactions. TnsA mediates DNA cleavage reactions at the 5' ends of Tn7, and TnsB mediates DNA breakage and joining reactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result from a collaboration between two active sites, one in TnsA and one in TnsB; the same (or a closely related) active site in TnsB also mediates the subsequent joining of the 3' ends to the target. Both TnsA and TnsB appear to be members of the retroviral integrase superfamily: mutation of their putative DD(35)E motifs blocks catalytic activity. Recombinases of this class require a divalent metal cofactor that is thought to interact with these acidic residues. Through analysis of the metal ion specificity of a TnsA mutant containing a sulfur (cysteine) substitution, we provide evidence that a divalent metal actually interacts with these acidic amino acids.
Sarnovsky
R J
RJ
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
May
E W
EW
Craig
N L
NL
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000737:chemistry*; D004251:DNA Transposable Elements / Q000235:genetics; D004278:DNA, Superhelical / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D019426:Integrases / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D012190:Retroviridae / Q000201:enzymology; D016415:Sequence Alignment; D019895:Transposases
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D008345:Manganese; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D019895:Transposases; D003545:Cysteine