The integrase of the conjugative transposon Tn916 directs strand- and sequence-specific cleavage of the origin of conjugal transfer, oriT, by the endonuclease Orf20

Abstract

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.

FIG. 1.

Cleavage of Tn916 oriT_200-2 by MBP-Orf20. The left panel shows cleavage of the T strand. Lanes 1 to 4: DNA-sequencing reactions. Lane 5: DNA probe alone. Lanes 6 to 9: MBP-Orf20 increasing from 37.5 nM to 1.25 μM. The right panel shows cleavage of the NT strand. Lane 1: DNA probe alone. Lanes 2 to 5: MBP-Orf20 increasing from 37.5 nM to 1.25 μM. Lanes 6 to 9: DNA-sequencing reactions.

FIG. 2.

Map of oriT_200-2. The TGGT motif identified as nic is underlined, with the cleavage site indicated by a black downward pointing arrow. MBP-Orf20 nonspecific cleavage sites are identified by thin upward pointing arrows. The Int binding site is indicated by a dashed line above the sequence. Inverted arrow pairs indicate inverted repeats identified by Jaworski and Clewell (14). Instances of GT at cleavage sites are boldface.

FIG. 3.

Effect of Int on cleavage by MBP-Orf20. Left panel: T strand. Right panel: NT strand. Lane 1: DNA probe in the presence of 320 nM Int. Lane 2: 125 nM MBP-Orf20. Lanes 3 to 6: 125 nM MBP-Orf20 with increasing amounts of Int from 40 nM to 320 nM. Lanes 7 to 10: DNA-sequencing reactions.

FIG. 4.

Cleavage by MBP-Orf 20 leaves a 3′ OH group. Lane 1: DNA alone. Lane 2: DNA plus 160 nM Int. Lane 3: DNA plus 125 nM MBP-Orf20. Lane 4: DNA plus 160 nM Int plus 125 nM MBP-Orf20. Lanes 5 to 8: DNA sequence ladder. The asterisk indicates the specific cleavage product produced in the presence of MBP-Orf20 and Int.

FIG. 5.

Absence of cleavage of a mutant DNA template by MBP-Orf20. Lane 1: DNA plus 320 nM Int. Lane 2: 125 nM MBP-Orf20. Lanes 3 to 6: 125 nM MBP-Orf20 plus increasing amounts of Int from 40 nM to 320 nM. Lanes 7 to 10: DNA sequence ladder. The position of the sequence alteration from TGGT to GTTG is indicated.

FIG. 6.

Alignment of Tn916 Orf20 and related proteins. The proteins are separated into two groups based on their similarity to Tn916 Orf20. The first group (group I) includes those proteins scored as at least 63% identical and 82% similar to Orf20 by BLASTP (1) analysis. The second group (group II) is comprised of proteins scoring less than 40% identical and 59% similar by BLASTP. Alignments were generated by CLUSTAL W (35). Markings below the alignments indicate degrees of similarity between the sequences: *, single fully conserved residue (group I); :, conservation of strong groups (group I); · , conservation of weak groups (group I); !, single fully conserved residue (all sequences); ‡, conservation of strong groups (all sequences); †, conservation of weak groups (all sequences); no mark, no conservation. Highly conserved tyrosines among all proteins are shaded light gray. The one completely conserved tyrosine is indicated by white letters on a black background. Residues implicated in the modified 3-H motif are shaded dark gray. The proposed motifs (based on Rep protein motifs assigned by Koonin and Ilyina [15]) are indicated above the sequences. The protein sequences shown and their accession numbers are as follows: Orf20 of E. faecalis Tn916, AAB60013; Orf20 of C. difficile Tn5397, AAO24811; Enterococcus faecium DO putative replication initiation factor protein EfaeDRAFT_2437, ZP_00603105; E. faecalis V583 Cro/CI family protein EF1886, AAO81639; Listeria monocytogenes EDG-e Orf20 homolog lmo1111, AG1213; Staphylococcus aureus Mu50 putative phage replication protein SAV0408, BAB56570; S. aureus COL replication initiation factor family protein SACOL1583, AAW38199; S. aureus MRSA252 conserved hypothetical protein SAR1297, CAG40299; B. subtilis 168 transposon-like protein YdcR, CAB12294; Geobacillus stearothermophilus cryptic plasmid pSTK1 Orf3, 2102242C; Streptococcus thermophilus putative transfer protein OrfJ, CAE52362; and Streptococcus mutans UA159 putative transposon protein SMU.207c, AAN57979.