Communications between distant sites on supercoiled DNA from non-exponential kinetics for DNA synapsis by resolvase¹

Abstract

To determine how distant sites on supercoiled DNA communicate with each other, the mechanism of site-specific recombination by resolvase was analysed by using a rapid-reaction quench-flow device to study the kinetics of individual steps in the reaction pathway. Three sets of measurements revealed the rates for: (1) the initial binding of the protein to its target sites on the DNA; (2) the synapsis of the two DNA-protein complexes; (3) the overall process of recombination. The binding of the protein to the DNA was complete within 50 milliseconds while recombination required 500 seconds. Surprisingly, synapsis spanned this entire time range: some DNA molecules gave synaptic complexes within ten milliseconds after the initial binding, while others took over 100 seconds. The departure from exponential behaviour may be due to each molecule of DNA having to undergo different conformational fluctuations in order to juxtapose the recombinational sites. From polymer physics theory, the rate of synapsis ought to vary with either the size of the DNA molecule or the length of DNA between the recombinational sites, depending on the nature of the fluctuations, but plasmids of different sizes and with different spacings between the sites all gave the same rates for synapsis. This observation cannot be reconciled with current models for encounters of distant sites on supercoiled DNA. However, the superhelical axis in the DNA molecules used here will be branched at one or more positions and the encounters may arise from the motion of a single branch relative to the remainder of the chain. Alternatively, the non-exponential kinetics for synapsis may be due to multiple re-arrangements of non-productive complexes following DNA juxtaposition.

Introduction

Many genetic events require communications between proteins bound to distant sites on DNA. Examples include DNA replication, gene expression and its control, site-specific recombination and other genome re-arrangements Gellert and Nash 1987, Bellomy and Record 1990, Schleif 1992. The communications occur more readily when the two sites are in cis, on the same DNA molecule, than with sites in trans, on separate molecules, principally because the local concentration of one site in the vicinity of another will be higher when both sites are on the same chain (Rippe et al., 1995). The local concentration of two sites on the same chain is increased further by supercoiling, since a supercoiled DNA will occupy a smaller volume than a relaxed DNA (Vologodskii & Cozzarelli, 1994). Consequently, many systems for long-range interactions between distant sites operate efficiently only with sites in cis on supercoiled DNA, though some reactions spanning distant sites are additionally influenced by supercoiling for other reasons Nash 1990, Kanaar and Cozzarelli 1992. Of the many genetic processes that require two sites on a supercoiled DNA, one of the best characterised is site-specific recombination by resolvase Grindley 1994, Oram et al 1995. This study describes a new approach to the analysis of long-range interactions on DNA, by correlating the kinetics of recombination by Tn21 resolvase to the dynamics of DNA fluctuations from polymer physics. The following paper (Sessions et al., 1997) describes a numerical model to account for the kinetics of DNA synapsis reported here.

Resolvase is a protein encoded by Tn3-like transposons that mediates part of the transposition process for these elements, the recombination event between the res sites of the transposon (Hatfull & Grindley, 1988). In vitro, resolvase converts a circular DNA with two res sites into two circles of DNA (Reed, 1981), interlinked once to form a catenane of unique topology (Wasserman & Cozzarelli, 1985), provided that the DNA is negatively supercoiled and that the res sites are oriented in direct repeat (Stark et al., 1989). These reactions were first characterised with the resolvases from Tn3 and γδ (Hatfull & Grindley, 1988) though the resolvases from other Tn3-like transposons, such as Tn21, function similarly. However, they possess distinct specificities for the DNA sequences at their respective res sites (Avila et al., 1990). The res sites from both Tn3 and Tn21 are ∼120 bp long and have three separate binding sites for the respective resolvases, named I, II and III Grindley et al 1982, Rogowsky et al 1985, Hall and Halford 1993. Nevertheless, Tn21 resolvase has no activity at Tn3 sites nor vice-versa (Ackroyd et al., 1990).

The pathway for recombination by resolvase has three main stages: first, the initial binding of the protein to the DNA; second, the formation of a synaptic complex in which the DNA-protein complexes at the two res sites interact with each other; third, the strand transfer reactions. The latter includes the cleavage of specific phosphodiester bonds in both strands of the DNA at both res sites, with each terminus becoming covalently linked to a serine at the active site of the protein, the alignment of these termini to their partners from the other res site and, finally, the re-ligation reactions (Stark et al., 1992). However, the first and second stages also involve numerous steps. The binding of resolvase to res is not a simple 1:1 association but rather a series of associations. Though the cross-over occurs specifically at site I, all three binding sites at both res sites have to be loaded with the dimeric form of the protein before the reaction can proceed Hughes et al 1993, Soultanas et al 1995. Moreover, all of the steps in the strand transfer reaction occur within a synaptic complex that incorporates sites I, II and III from both res sites Reed and Grindley 1981, Grindley 1994.

The simplest mechanism for forming a complex between two sites on a DNA chain is that the sites meet each other by unconstrained random collisions stemming from the dynamic flexibility of the chain. On a circle of supercoiled DNA, some encounters of this type would trap a large number of supercoils between the sites and others fewer supercoils, so a recombination reaction across the sites that sequesters the intervening DNA into a separate circle would lead to DNA catenanes with variable numbers of interlinks between the two product rings (Wasserman & Cozzarelli, 1986). For many reactions that span distant sites, the sites do indeed come together by random collisions: examples include site-specific recombination by λ integrase and DNA cleavage by the SfiI endonuclease Gellert and Nash 1987, Szczelkun and Halford 1996. However, resolvase produces only singly interlinked catenanes and this particular topology demands that just three negative supercoils are located between the synapsed res sites (Wasserman & Cozzarelli, 1985; Stark et al., 1989). In a productive synapse, these supercoils are fixed by the plectonemic wrapping of the two DNA segments around each other and around the protein Benjamin and Cozzarelli 1988, Benjamin and Cozzarelli 1990, Stark et al 1994.

Two mechanisms have been proposed for DNA synapsis by resolvase (Figure 1). In one, from Boocock et al. (1986), the sites meet by random collisions in any geometry but only a fraction of the encounters yield synaptic complexes with three trapped supercoils and only these proceed directly into the recombination reaction: the remainder of the encounters produce complexes with inappropriate geometries and these presumably dissociate before trying again (Figure 1a). On this scheme, the average DNA molecule will undergo many rounds of random collisions before alighting on the correct structure. In the other, called “slithering” (Benjamin & Cozzarelli, 1986), a reptational motion of the DNA chain around a fixed superhelical axis leads directly to complexes with three supercoils between the res sites (Figure 1b). Slithering might appear to be the faster route to the correct synapse (Parker & Halford, 1991) but hydrodynamic considerations indicate that this is not so, at least for DNA of >2 kb (Marko & Siggia, 1994). From polymer physics theory (Marko & Siggia, 1995), the juxtaposition of two sites on a supercoiled DNA of 4 kb ought to take about one-second by slithering but only ∼50 ms by random collisions.† Even so, this difference is insufficient to distinguish the models, since 20 or more random collisions may be needed to generate a productive complex. Nevertheless, the two models can be distinguished by altering either the size of the DNA molecule or the length of DNA between the res sites. On an unbranched DNA with a single superhelical axis, the time constant for synapsis by slithering ought to increase steeply as the overall length of the DNA is increased but it will be virtually independent of the length of DNA separating the sites, since synapsis can be achieved whenever the mid-point between the sites reaches the apex of the superhelix Marko and Siggia 1994, Marko and Siggia 1995. Random collisions should show the opposite behaviour: a marked dependence on the length of DNA between the sites, since this will determine the local concentration of one site in the vicinity of the other, but no significant dependence on the overall length of the chain (Berg, 1984).