Homology-dependent interactions determine the order of strand exchange by IntDOT recombinase

Abstract

The Bacteroides conjugative transposon CTnDOT encodes an integrase, IntDOT, which is a member of the tyrosine recombinase family. Other members of this group share a strict requirement for sequence identity within the region of strand exchange, called the overlap region. Tyrosine recombinases catalyze recombination by making an initial cleavage, strand exchange and ligation, followed by strand swapping isomerization requiring sequence identity in the overlap region, followed by the second cleavage, strand exchange and ligation. IntDOT is of particular interest because it has been shown to utilize a three-step mechanism: a sequence identity-dependent initial strand exchange that requires two base pairs of complementary DNA at the site of cleavage; a sequence identity-independent strand swapping isomerization, followed by a sequence identity-independent cleavage, strand exchange and ligation. In addition to the sequence identity requirement in the overlap region, Lambda Int interactions with arm-type sites dictate the order of strand exchange regardless of the orientation of the overlap region. Although IntDOT has an arm-binding domain, we show here that the location of sequence identity within the overlap region dictates where the initial cleavage takes place and that IntDOT can recombine substrates containing mismatches in the overlap region so long as a single base of sequence identity exists at the site of initial cleavage.

Figure 1.

(A) Wild-type sequences of the core-binding sites attDOT and attB. The boxed region indicates the overlap region (o). The conserved GC dinucleotide is shown at the left sides of the overlap regions. The remaining bases in the overlap are the coupling sequences that vary from site to site. The small arrow represents orientation of the overlap region relative to flanking arm sites. The longer arrows indicate the imperfect inverted repeats that flank the overlap regions. The D and B sites contain 8 of 10 identical base pairs. The vertical arrows show the sites of cleavage on the top and bottom strands. (B) Simplified schematic diagram of the same core regions. The bubbled area denotes the GC dinucleotide homology between the attDOT and attB1 overlap regions. (C) Schematic diagram of the attB site with an inverted overlap region. The arrow denotes the directionality of the overlap sequence. (D) Schematic diagram of the attB site containing a symmetric overlap region. The line over the ‘o’ represents a line of symmetry within the overlap region. (E) Sequences of the wild type, inverted and symmetric overlap regions.

Figure 2.

Schematic model of how the plasmid containing the attDOT site orients itself relative to the attB to maintain alignment of the GC dinucleotide. (A) Orientation of wild-type attDOT and wild-type attB in the standard integration reaction. The vertical arrow indicates the site of initial cleavage. (B) Possible orientation of wild-type attDOT and the attB site containing an inverted overlap region. (C) Products from the attDOT and attB sites oriented as shown in B if the initial cleavage occurs at the location of the vertical arrow. There is complete heterology within the entire seven base overlap region in both products. (D) Rotation of the inverted overlap attB site by 180° orients the GC dinucleotide in the same position as in the wild-type reaction. The vertical arrow denotes the site of initial cleavage producing the recombinants that both contain the complementary GC dinucleotide on the left side.

Figure 3.

(A) Schematic representation of the in-vitro integration assay and the single asymmetric SspI site that produces 1.1 and 2.5 kb fragments from the linear recombinant. (B) Results from an SspI digest of recombinants produced from wild-type attDOT and either wild-type or inverted overlap attB sites. A T for the top strand or a B for the bottom strand indicates the strand that is radiolabeled.

Figure 4.

(A) Recombination reactions with attB suicide substrates nicked on either the top or bottom strand. The attB substrates contain a nick at either the top (right) or bottom (left) strand at the cleavage site. Initial strand exchange can occur between the intact strand of attB and the corresponding strand of attDOT, allowing determination of strand exchange order. The strand containing a nick cannot undergo recombination and so traps a Holliday intermediate where the first strand has been exchanged but the nicked strand has not. (B) Integration assay results using wild-type attDOT and either wild-type attB; inverted overlap intact attB; inverted overlap attB containing a nick in the top strand; or inverted overlap attB containing a nick in the bottom strand. Each sample is run in duplicate on the gel. (C) Graph showing the average percent recombination of the intact and nicked attBs over a minimum of four experiments.

Figure 5.

(A) SspI restriction digest of recombinants produced from either wild-type attB1 or attB containing a symmetric overlap sequence. (B) Graph comparing the average percent recombination of wild-type attB1 and an attB site containing a symmetric overlap region. All attB sites were combined with a wild-type attDOT and averaged over a minimum of four experiments.

Figure 6.

(A) Sequence of BoB attB mutants with each core overlap sequence. The B region of the core is normally only on the 5′ side. In the BoB mutant, it is present on both sides. In this study, they are combined with wild-type attDOT containing both the D and D’ core-type sites. (B) Comparison of the average percent recombination of wild-type attDOT combined with wild-type (WT BoB’), inverted overlap (Inv. BoB’), or symmetric overlap attB1 (Sym. BoB’). And wild-type attDOT combined with BoB attB sites containing wild-type (WT BoB), inverted overlap (Inv. BoB) or symmetric overlap (Sym. BoB) regions. The average recombination was taken over a minimum of four experiments.

Figure 7.

(A) Sequences of attDOT and attB1 with wild-type or inverted overlap regions. (B) Integration assay results from combinations of wild-type (WT) and inverted overlap (Inv.) attDOT and attBs. The average recombination was taken over a minimum of four experiments.

Figure 8.

(A) Sequence comparison of wild-type attDOT and an attDOT site with a terminal G mutation in the overlap region, and wild-type attB with an attB site containing substitutions at both ends of the overlap region. (B) Sequences of products formed from the recombination of wild-type or TermG attDOT with either wild-type or AT attB. The second pair of sequences in each combination is the products from an inverted attB site. Complementary base pairs are in bold. (C) Integration assay results from combinations of wild-type or TermG attDOT sites with either wild-type or AT attB sites. The average recombination was taken over a minimum of four experiments.