Orf20 of the conjugative transposon Tn916 was purified as a chimeric protein fused to maltose binding protein (MBP-Orf20). The chimeric protein possessed endonucleolytic activity, cleaving both strands of the Tn916 origin of conjugal transfer (oriT) at several distinct sites and favoring GT dinucleotides. Incubation of the oriT DNA with purified Tn916 integrase (Int) and MBP-Orf20 resulted in strand-and sequence-specific cleavage of oriT at a TGGT motif in the transferred strand. This motif lies immediately adjacent to a sequence in oriT previously shown to be protected from DNase I cleavage by Int. The endonucleolytic cleavages produced by Orf20 generated a 3 OH group that could be radiolabeled by dideoxy ATP and terminal transferase. The production of a 3 OH group distinguished these Orf20-dependent cleavage events from those catalyzed by Int at the ends of Tn916. Thus, Orf20 functions as the relaxase of Tn916, nicking oriT as the first step in conjugal DNA transfer. Remarkably for a tyrosine recombinase, Tn916 Int acts as a specificity factor in the reaction, conferring both strand and sequence specificities on the endonucleolytic cleavage activity of Orf20.Tn916 is an 18-kb conjugative transposon originally discovered in Enterococcus faecalis DS16 (9). This element confers tetM tetracycline resistance on its host (8), catalyzes its own excision and integration (17, 25), and carries conjugal transfer functions, orf13-orf24, in its tra region (30). It is a highly promiscuous element able to transfer between different species and genera of gram-positive organisms as well as gram-negative bacteria, such as Escherichia coli and Pseudomonas fluorescens (6, 24). Conjugative transposons have been shown to mediate transfer of antibiotic resistance in complex microbial communities, such as the intestinal flora (31).Transposition of Tn916 begins with an excision event catalyzed by the transposon-encoded integrase (Int) and Xis proteins (17, 25), resulting in the formation of a circular, nonreplicative intermediate. Genetic evidence suggests that a single strand of the intermediate is then transferred to the recipient (28), where it undergoes replication and subsequent integration into the new host's genome (33). The segment of Tn916 designated the origin of conjugal transfer (oriT) lies in the noncoding region between orf20 and orf21 (14). This 500-bp DNA segment can catalyze in cis the mobilization of a nonconjugal plasmid by Tn916. The sequence of oriT possesses a number of inverted repeats and sequences that are similar to origins of transfer in IncP and F-like plasmids. Certain regions within oriT were postulated to be nic sites, where transfer is initiated by cleavage by a putative endonuclease/relaxase and the formation of a protein-DNA complex that serves as a substrate for DNA transfer (the relaxosome) (14). However, neither the exact location of nic nor the protein responsible for cleavage within oriT has been identified.The broad-host-range nature of Tn916 and comparisons with other bacterial conjugation syst...

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