Synapsis and strand exchange in the resolution and DNA inversion reactions catalysed by the beta recombinase

Abstract

In the presence of a sequence-independent chromatin-associated protein, such as Hbsu or HMGB, the beta recombinase catalyses resolution between two directly oriented recombination sites (six sites) and both resolution and DNA inversion between two inversely oriented six sites. Assembly of the synaptic complex requires binding of the beta recombinase to the six sites and the presence of Hbsu. Whether resolution or inversion will take place depends on the relative orientation of the two six sites, the level of DNA supercoiling and the amounts of Hbsu. In this work, the topologies of the products of the resolution and inversion reactions were analysed. The resolution reaction generated mainly singly catenated DNA circles, while DNA inversion gave rise to unknotted circles and small amounts of DNA molecules containing 3- or 5-noded knots. In spite of the distinctive features of the beta system, the topology of synapsis and strand exchange during the resolution reaction is similar to that of Tn3 and gammadelta resolvases. The ability of the beta recombinase to catalyse both inversion and resolution reactions probably reflects different possible architectures of the synaptic complex, which to a large extent depends on Hbsu.

Figure 1

Recombination substrates used. (A) Plasmids pCB8 and pCB12, containing two recombination sites (six sites) in either direct or inverse orientation, respectively, are represented. The six sites are depicted as open and filled arrows. Restriction sites relevant for the analysis of the recombination products are indicated. (B). Orientation and sequence of the six sites present in the plasmids used in this work. Each six site is composed of two subsites, named I and II, which are binding sites for the β recombinase. Subsite I has a dyad symmetry and is represented by arrows. Subsite II has no obvious symmetry and is represented by an open box. The length of the DNA segments (in bp) is indicated in Arabic numbers. The sequence of the crossover site, which is located at the centre of subsite I, is shown for each six site of the plasmids used. Plasmid pCB8 and pCB12 have two identical wild-type six sites. Plasmids pCB81 and pCB141 have one copy of a wild-type six site and another copy of a six site with an A→C substitution at the crossover site. The relative orientation of each site is indicated with open or filled arrows.

Figure 2

Topology of the resolution products of β recombinase. The β recombinase (50 nM) and Hbsu (50 nM) proteins were incubated with either pCB8 or pCB81 (10 nM) for 30 min at 37°C. (A) The substrates (lanes 1 and 3) and the reaction products (lanes 2 and 4) were digested with SalI and PstI and separated in a 0.8% agarose gel. Lin, linear product; NR, non- recombinant molecules; RP, resolution products. The small 0.5 kb SalI DNA segment is not shown. (B) The different topoisomers present in the undigested substrates (lanes 1 and 3) and recombination products were resolved in 0.8% agarose gels (lanes 1–4 and 7–10), in parallel with a ladder of topoisomers obtained by partial treatment of plasmid pCB8 with topoisomerase II (lanes 5 and 6). In lanes 7–10, the DNA was nicked by a partial treatment with DNase I. Numbers on the right indicate the migration of knots or catenanes containing 0, 2, 4 or 8 nodes; sc denotes the position of supercoiled DNA. (C) Predicted topology of the reaction products if three supercoils are trapped. One round of recombination (180°) would generate a 2-noded catenane. A second round of strand exchange (360°) would generate a non-recombinant 4-noded knot. Based on Stark et al. (28).

Figure 3

Topology of the reaction products obtained with a supercoiled or relaxed plasmid containing two inversely oriented six sites. The β recombinase (50 nM) and Hbsu (300 nM) proteins were incubated with either pCB12 or pCB141 (10 nM) for 90 min at 37°C, the substrates and reaction products digested with KpnI and EcoRV and separated in a 0.8% agarose gel. (A) The supercoiled substrates (lanes 2 and 4) and the reaction products (lanes 3 and 5) were digested and separated. (B and C) The different species present in the undigested substrates and recombination products were resolved in 0.8% agarose gels in parallel with a ladder of topoisomers obtained by partial treatment of plasmid pCB12 with topoisomerase II (lanes 7 and 8 in panel B and lane 5 in panel C). Where indicated, the DNA was nicked by a partial treatment with increasing concentrations of DNase I. (D) The relaxed substrate (lane 2) and the reaction product (lanes 3) were digested with KpnI and EcoRV and separated in a 0.8% agarose gel. (E) The topoisomers present in the undigested substrates and recombination products were nicked by a partial treatment with increasing concentrations of DNase I (lanes 1–4) and resolved in 0.8% agarose gels in parallel with a ladder of topoisomers obtained by partial treatment of plasmid pCB12 with topoisomerase II (lane 5). Lin, linear product; IP, inversion products; NR, non-recombinant molecules; RP, resolution products. Numbers on the right of the figure indicate the migration of topoisomers containing 0, 2, 3, 4 or 5 supercoils; sc and lin denote the position of supercoiled and linear DNA, respectively.

Figure 4

Proposed models for β-mediated recombination in the presence of Hbsu. (A and B) The two six sites for the β recombinase are represented. The binding sites for β recombinase are indicated with filled arrows (subsite I) or filled boxes (subsite II). The length of the DNA segments (in bp) is indicated in Arabic numbers. The β recombinase dimers, which interact differently with subsites I and II (see 22), are represented with filled spheres. The horizontal arrows above the six site denote the relative orientation of the recombination sites. In (A)–(D) the lower case letters are used to position the respective subsite in the synaptic complex. Speculative models for synaptic complexes containing β recombinase and Hbsu are presented for DNA substrates containing two six sites in direct orientation (C) or in inverse orientation (D). The large spheres correspond to β recombinase, while small spheres denote the Hbsu protein. The model for the resolution synapse shows the three trapped (–) supercoils, while that for the inversion synapse assumes that no supercoils are trapped.