Site-Specific Recombination: Integration, Excision, Resolution, and Inversion of Defined DNA Segments
Chapter
125
HOWARD A. NASH
Genetic stability is a cornerstone of life. But, against the background of genome stability, nature tolerates and occasionally favors a modest degree of variation. One kind of variation involves specific rearrangements of DNA. Such events are distinguished from other types of recombination in that they are focused at special sites in the genome. The concept of recombination between specific loci came from studies of the integration and excision of bacteriophage lambda. Allan Campbell’s proposal for these processes (12) had enormous success in guiding genetic experiments on the nature of the putative recombination sites and the genes that direct their reciprocal joining. These experiments (31) solidly established site-specific recombination as a paradigm for variation in the genome. Since the late 1970s, the emergence of techniques for physical examination and manipulation of DNA has led to the discovery of many more systems that exploit rearrangement at specific sites. This chapter first presents the features that these systems share with each other and the different ways they contribute to the biology of bacteria and accessory genetic elements such as bacteriophages, plasmids, and transposons. Later sections outline what is understood about the way specific recombinases promote these events and how accessory proteins assist some of these recombinases. Finally, the means of controlling the timing and efficiency of these recombination events will be surveyed and a few speculations on questions for future research will be presented.
One can think of many rearrangements of the genome as a genetic exchange between two partners. In a typical site-specific recombination, both partners carry a well-defined specific site that is necessary for the recombination event and that contains the point of genetic exchange. The degree to which these sites are specified dictates the uniqueness of the rearrangement. For example, in lambda integration the same point in the 46.5-kbp viral chromosome is involved in virtually all events and, even more impressively, in the vast majority of cases lambda inserts into a unique target in the 4.5-Mbp Escherichia coli genome. Similarly, in most of the cases discussed in this chapter the recombination loci of both partners are highly specified, and as a result, the rearrangement is uniquely defined. Exceptions to this rule provide an interesting set of variations that can be related to the main theme. Of course, recombination loci are not the only well-defined sites in E. coli and Salmonella typhimurium (official designation, Salmonella enterica serovar Typhimurium). For example, origins of replication, sites of viral packaging, promoters, origins of chromosome mobilization, etc., are also highly specified. What distinguishes the loci of site-specific recombination is that they function not singly but in pairs. Nevertheless, studies of the defined targets of site-specific recombination and studies of other kinds of unique sites in the genome have frequently provided one another with technical and conceptual advances.
When considering a pair of recombination loci, a critical issue concerns the polarity of the sites. If both sites of the pair are polar and therefore can be described by an arrow having a head and a tail, recombination can join them in a unique way. Conversely, a nonpolar (functionally palindromic or symmetric) site cannot specify relative orientation. In naturally occurring site-specific systems, the recombination locus of both partners is polar. Thus, these systems have not only positional specificity but also orientational specificity, a feature that is often important for their biological function and one which raises interesting mechanistic questions.
What directs the recombination of these specialized sites? A formal possibility is that the sites are simply regions of homology between partners and are subject to the action of a homologous recombination system, such as that based on the RecA protein of E. coli or the Red proteins of bacteriophage lambda. However, this possibility has not been exploited in nature, and in virtually every case a specialized protein, a specific recombinase, is devoted to the task. For example, in the case of bacteriophage lambda, integration is found to depend on a gene, appropriately given the name int, that maps adjacent to the viral locus of integration. Similarly, many (but not all) of the site-specific recombinases are found to map adjacent to their site of action, an arrangement that may provide an efficient means for their evolution and distribution (10). Regardless of the location of the gene for the recombinase, regulation of its synthesis provides a way to control the timing and efficiency of the rearrangement that it promotes. Moreover, specific binding of the recombinase to the recombination locus is an important part of the mechanism for selecting the site.
The final feature that characterizes the site-specific systems is the "conservative" nature of the recombination event. The term "conservative" means that nothing is degraded or added during the recombination, a description that applies at several levels of molecular detail. First, no genetic information is lost or gained as a consequence of recombination; the sequence of the rearranged segments is simply a permutation of the parental DNA. A second conservative feature of site-specific recombination is the absence of replication. Not only is the genetic information of the parents retained in the recombinants, but the actual nucleotides that make up the parental DNA are conserved. Thus, site-specific recombination is the archetype of break-and-join recombination and has no contribution from copy-choice or break-copy mechanisms (94). This conservative feature distinguishes site-specific recombination from transposition, in which repair synthesis is integral to the mechanism and can be massive (chapter 124, this volume). Similarly, most models for homologous recombination call for significant DNA synthesis. A third level of conservation in site-specific recombination is thermodynamic. In every case tested, breakage and reunion take place without the intervention of high-energy cofactors (such as ATP), implying that the energy stored in the phosphodiester backbone of DNA is retained at every step of the rearrangement. As explained below, this follows from the fact that site-specific recombinases are topoisomerases and not nucleases.
As a consequence of their specific and conservative nature, one can think of site-specific recombination systems as precise and delicate tools for rearranging the genome, a feature exploited both in nature and, increasingly, by genetic engineers.
The capacity to generate precise rearrangements has been exploited widely in E. coli and S. typhimurium. To organize a large and rapidly growing body of knowledge on the uses of site-specific recombination, it is convenient to group these systems by the structural consequences of their action. Figure 1 contrasts three possible ways that two polar sites may be distributed in a genome. Each site can be situated on a separate element of the genome, or the sites can be situated on the same element, either in a head-to-head or head-to-tail configuration. In the first case, integration (Fig. 1A), recombination joins the two elements into a single unit. (Recombination between loci on separate linear elements or between a linear and a circular element is also possible and has obvious structural consequences.) In the second case, inversion (Fig. 1B), recombination flips one segment of the element with respect to the other, while in the final case, resolution or excision (Fig. 1C), recombination splits one element into two.
How are these different arrangements of recombination loci used to produce biologically meaningful outcomes? The best-known examples come from systems in which this kind of recombination was first discovered. For example, the Campbell scheme (12) for recombination between separate genetic elements (Fig. 1A) shows how a genetic element (the circular chromosome of E. coli) acquires foreign genetic information (the circular chromosome of bacteriophage lambda). The result is a lysogen, an E. coli bearing an integrated lambda chromosome (prophage).
The two systems that epitomize site-specific inversion (Fig. 2) are used to generate diversity in gene expression. Inversely oriented recombination loci flank a promoter of the flagellar control region of S. typhimurium (96). Recombination flips the promoter segment to orient it toward or away from important structural and regulatory genes (Fig. 2A). These two alternative arrangements of the control region neatly account for the phase variation in S. typhimurium, a phenomenon discovered in the 1920s and shown by genetic experiments in the 1950s to be linked to a "heritable variation" in the control region (52). In a similar way, inversely oriented recombination sites encompass two open reading frames that encode alternate carboxy termini for a tail fiber gene of bacteriophage Mu (Fig. 2B). Recombination positions one or the other of these open reading frames adjacent to a common amino-terminal segment and thereby provides the phage with two alternative gene products that direct two different host ranges (29).
Recombination between two directly repeated loci (Fig. 1C) is the way that a lambda lysogen regenerates a free lambda chromosome and a cured E. coli chromosome (12). Recombination of directly repeated loci also occurs during transposition of the class of mobile elements exemplified by Tn3. Here, transposition involves the concerted act of replicating the element and joining it to a new target (Fig. 3). When the transposon starts in one circular element and hops to another, the result is a chimera, called a cointegrate, in which the two parental circles are joined and a copy of Tn3 is present at each of the two join points. The fusion of the two circular elements reduces the independence of each copy of the transposon; i.e., they cannot spread to independent hosts. However, in addition to the signals that direct its replicational transposition, Tn3 also contains a site-specific recombination system (3). Recombination between the specific locus in each copy of the transposon resolves the cointegrate into two circular elements, each of which has a new copy of Tn3 (Fig. 3).
These few biological functions—acquisition and elimination of defined segments of DNA, generation of diversity in gene expression, and reduction of dimeric forms—appear to be the raison d’être for an impressive array of different site-specific recombination systems. A survey of such elements is presented in Table 1. To provide a sense of the scope of site-specific recombination in the bacterial world, the table is not limited to systems known to operate in E. coli and S. typhimurium. In the table each recombination system is assigned a function, but the reader should be aware that in some cases this has not been proven. Moreover, even when biological experiments have clearly established that recombination performs the indicated function, it is possible that the same recombination components can be used for additional purposes; the listed functions are not exclusive. Even within a given functional grouping, there are important variations. For example, generation of diversity in gene expression can either utilize inversely repeated sites, as described in Fig. 2, or directly repeated sites, as in the removal of segments of DNA that interrupt genes in Bacillus subtilis (85) and Anabaena variabilis (49). Another variation involves the complexity of DNA that undergoes rearrangement. For integration and excision into and out of the bacterial chromosome, the elements range in size from bacteriophage genomes (lambda), through plasmids (pSAM2) and nonduplicative transposons (Tn916), to single genes (integrons).
Table 1Site-specific recombination systems. |
Of particular interest are systems that exploit site-specific recombination to reduce dimeric forms, because of their potential role in each bacterial generation. In any replicating system, dimers can arise by homologous recombination between sister chromosomes. At the time of cell division, segregation of these dimers to one daughter or the other can severely imbalance the orderly inheritance of the affected element. Bacteriophage P1 encodes a site-specific recombination system (cre) that is needed to insure the faithful maintenance of this low-copy circular element, apparently by reducing these accidental dimers (4). Similarly, plasmids of the ColE1 family exploit an E. coli-encoded site-specific recombination system (xer) to resolve multimeric forms and enhance the efficiency of segregation (87). The xer system is apparently also used in the same way at a locus (dif) in the region of the E. coli genome that is devoted to the termination of replication (5, 55a). In this sense, at every generation site-specific recombination operates on the entire E. coli genome. Deletion of the dif locus or inactivation of the Xer recombinase leads to aberrations in cellular partitioning of daughter chromosomes, induction of the SOS response, and filamentation. Thus, site-specific recombination is a major contributor to the fitness of E. coli.
Any conservative site-specific recombination is likely to involve several mechanistic steps that are at least conceptually distinct. The DNA of two recombination loci must first be recognized. In a step called synapsis, the loci must then be brought into physical contact. After cleavage of the synapsed loci, the broken ends must then be transferred to new partners and rejoined. For several of the systems described above, the recombination pathway has been analyzed at genetic and biochemical levels. Using this powerful combination of approaches, workers have dissected the recombination loci into their component parts, identified the gene products responsible for interacting with these parts, and established the principles by which the resulting protein-DNA assemblies effect a genetic rearrangement. Remarkably, despite considerable diversity in the function, complexity, and evolutionary history of these recombination systems, a common theme has emerged. At the heart of each recombination system is a 20- to 30-bp segment of the recombination locus, which is the "functional core" of the recombination site. Typically, this segment contains two binding sites, arranged as an inverted repeat, for a recombinase protein. Each of the elementary steps of site-specific recombination involves the DNA of the functional core and its bound recombinase. First, specific recognition of the core by the recombinase is a major factor in the fidelity with which DNA segments are singled out as targets for rearrangement. Second, pairs of these recombinase-core DNA complexes interact to form the synaptic structure. Finally, enzymatic activities of the bound recombinase protomers catalyze cleavage of core DNA and, after suitable realignment of the broken ends, ligation to the recombinant configuration. Although all site-specific systems share this theme, distinctions between different systems emerge from the particular way the elementary steps are carried out. Because they involve covalent change, the steps involving breakage and reunion of DNA have been the most completely characterized and are therefore most useful as distinguishing features.
The cleavage step of recombination involves attack by a recombinase polypeptide on a phosphodiester bond. Recombination requires four such cleavages, i.e., breakage of both strands in the cores of two partners. Each cleavage can be described as a reaction in which a phosphodiester bond linking 5' and 3' segments of a DNA strand (Fig. 4A) is replaced by a phosphodiester bond between a nucleophilic residue of the protein and one segment; in the process, the other segment is liberated (Fig. 4B). Cleavage is therefore not by hydrolysis of the phosphodiester, as would be catalyzed by a nuclease (Fig. 4E and F), but by transesterification with the recombinase.
On the basis of the details of strand cleavage, virtually all the site-specific recombination systems of Table 1 can be grouped into just two families. On the one hand are the recombinases, typified by lambda integrase, that use a tyrosine hydroxyl as the attacking nucleophile and that liberate a segment terminated by a 5' OH (19, 67). In all these systems, cleavage of one strand of the core is 6 to 8 bp to the 5' side of the cleavage point of the complementary strand (Fig. 5, left). On the other hand are the recombinases, typified by the enzymes of the resolvase and invertase systems, that use a serine hydroxyl as the attacking nucleophile and that liberate a segment terminated by a 3' OH (34, 46). In these systems cleavage of one strand is 2 bp to the 3' side of the cleavage point of the complementary strand (Fig. 5, right). The polypeptide sequences of the recombinases within any one family can be aligned, although there can be very substantial divergence in amino acid sequence (1, 2, 35, 76), indicative of an ancient common origin. However, significant similarities are not detected between members of the two different families, suggesting they have arisen independently.
To effect a genetic rearrangement, the cleaved strands from two partners must exchange positions with respect to each other. The details of such strand transfer are not known for any system, but topological experiments (19a, 41, 48, 64, 81a, 84) indicate that ordered movement is the rule and that there are strong controls on the kinds of movement that can occur. Ligation of transferred strands is the converse of the cleavage step: the phosphodiester linking the recombinase protein to one DNA segment is attacked by a hydroxyl residue from another segment of DNA, a segment created by prior cleavage of a partner strand (Fig. 4C). This transesterification liberates the recombinase from its covalent attachment to DNA and creates a new phosphodiester bond that joins segments of two parental DNA strands (Fig. 4D). No exogenous source of chemical energy is required for the rejoining. This is unlike the joining of DNA segments created by nuclease action, in which DNA ligase must use a high-energy cofactor to convert a DNA phosphomonoester into a diester (Fig. 4G and H). In the rejoining step of site-specific recombination, one kind of DNA phosphodiester merely replaces another.
Thus, at the chemical level, breakage (Fig. 4A and B) and reunion (Fig. 4C and D) follow the same pathway that was first described by James Wang for topoisomerase enzymes (reviewed in reference 26). Indeed, many proteins that have been identified by their DNA-relaxing or DNA-supercoiling activities can join strands from two partners and thereby create a recombinant molecule. What distinguishes the topoisomerase function of site-specific recombinases is that they are highly efficient at such strand exchange, presumably because they have the capacity to juxtapose partners prior to cleavage and to encourage strand transfer prior to ligation. In contrast, general topoisomerases usually rejoin broken DNA so as to reconstitute the original pattern of connectivity. For these enzymes, the important changes between the breakage and reunion steps are purely topological; strand exchange appears to be an uncommon, perhaps accidental, occurrence. In contrast, site-specific recombinases alter both the topology and the connectivity of their substrates.
A complete recombination event requires that both strands of each of two cores undergo breakage and subsequent ligation. One can imagine two extreme ways to couple these events. On the one hand, cleavage of top and bottom strands of a single core can be concerted, generating a double-strand break (Fig. 6B). In this case, recombination involves the rejoining of two partners, each of which has been completely disrupted (Fig. 6C). On the other hand, cleavage of one strand may precede that of the other (Fig. 6E). In this case, the initial exchange between partners involves transfer of a single strand. This creates an intermediate form (Fig. 6F), first proposed by Robin Holliday and accordingly called a Holliday junction, which contains two parental strands and two recombinant strands. Recombination is completed, i.e., the Holliday junction is resolved, when the remaining strand from each parent undergoes cleavage, transfer, and ligation (Fig. 6H). Where studied, all the members of the resolvaseinvertase family generate double-strand breaks and all members of the integrase family generate a Holliday junction intermediate.
Because there is a physical separation between the positions of exchange in the two strands of a recombination locus (Fig. 5), the products of recombination will contain a segment in which each strand is derived from a different parent (Fig. 6C and H). The segment, called the overlap region, is 6 to 8 bp long for different members of the integrase family and 2 bp long in all known members of the resolvaseinvertase family. The overlap region is a part of the recombination locus that is usually identical between partners. (The reader should be aware that the region of sequence identity between partners has often been called the core or, more specifically, the homologous core of a recombination locus. This need not be identical to the functional core, but the two usually share common elements.) Because of the identity of DNA sequence in the overlap region of the parents, the hybrid nature of the recombinant overlap region can typically be detected only by physical means. Although it is therefore normally genetically silent, the overlap region is an important determinant of site specificity through a requirement for sequence matching. For most systems, any alteration in the sequence of the overlap region of a recombination locus drastically reduces recombination frequency, but efficient recombination is restored if the identical substitution is introduced into both partners (6, 83, 92). Even multiple changes involving both transitions and transversions from the wild-type overlap sequence are permitted if the identical changes are introduced into the partner locus. Although changes in the length of the overlap region (75) and a few substitutions that apparently change DNA structure of the region (35, 90) are exceptions, the general rule seems to be that recombinases do not sense the sequence of the overlap region but instead sense the perfection of its match to a partner. In contrast, recombinase binding depends on sequences of the functional core that are located largely outside the overlap region. Here, natural or engineered differences between partners are tolerated (as long as an adequate binding site is maintained), showing that sequence matching is restricted to the overlap region. Such matching is usually described as demonstrating a requirement for homology between partners (92), but it must be emphasized that in homologous recombination this term implies only an approximation to identity, not the perfect matching required for site-specific recombination.
Biochemical studies have been useful in demonstrating that the block to recombination caused by nonhomology in the overlap region is only partial. The bulk of the evidence indicates that recombination initiates normally in these circumstances but cannot proceed to completion. For example, in an invertase system, when a circular DNA with a pair of nonhomologous recombination loci is treated with its cognate recombination enzyme, no recombinants accumulate, but the substrate shows topological changes (59). The details of these changes indicate that double-strand breaks have been made but that, instead of strands moving neatly to their recombinant configuration, more extensive strand motion is followed by rejoining of the broken recombination loci to reconstitute the parental connectivity. Similarly, in the lambda integrase family, partners with heterology in the overlap region form Holliday junctions but fail to resolve them so as to generate recombinant products (44, 65).
How do the recombinases sense that heterologous partners are attempting to recombine? It seems attractive to imagine that hybrid overlap regions are actually produced under these circumstances and that the mismatched base pairs engendered during recombination of heterologous partners abort the process (Fig. 7). For the resolvase/invertase systems, heteroduplex overlap regions would be made if nonhomologous loci not only suffer double-strand breaks but exchange strands and recombine. Although it has not been directly tested, these mismatched recombinants could be particularly good substrates for a new round of breakage, strand movement, and reunion; such a second cycle of strand exchange would reestablish parental connections (59, 83). For members of the lambda integrase family, movement of the Holliday junction across the overlap region is thought to occur by stepwise melting of parental duplexes, strand switching, and reannealing into hybrid configuration (92). This process, called branch migration, can move the Holliday junction from its point of synthesis toward its point of resolution 6 to 8 bp away (Fig. 6G). However, migration across a heterologous overlap region would result in mismatched base pairs, a distortion that should hinder the migration and prevent the branch from reaching its resolution point. Instead, the branch would drift back to its point of origin and be processed back to parental configuration. This view has recently become controversial as a result of experiments with artificial Holliday junctions formed from nonhomologous partners (1b, 20, 64a). However, experiments with a variety of other artificial recombination substrates (9a, 17, 63) provide strong suppport for the traditional view and prompt alternative explanations (9a) for the behavior of experiments with "frozen" junctions.
There are important exceptions to the rule that perfect homology is required in the overlap region of recombining partners (7, 13, 16, 56, 89). These cases are all members of the integrase family but have not yet been analyzed at the biochemical level; it will be of interest to see where the differences lie. One possibility concerns the stability of the Holliday junction. In systems that depend strongly on overlap homology, the Holliday junction is an unstable intermediate that either generates recombinants or is largely processed back to the parental configuration. For the systems that generate recombinants in vivo in the absence of identical overlap regions, the Holliday junction may be a stable product (58).
In general, naturally occurring overlap sequences are nonpalindromic. The asymmetric nature of this segment of the recombination locus stands in contrast to the remainder of the functional core, which comprises nearly or exactly inverted repeats for recombinase binding. The overlap segment is therefore an attractive candidate for the element that provides polarity to the recombination locus. Indeed, changing the overlap sequence in a pair of recombination targets for the bacteriophage P1 cre recombinase to perfect (and identical) palindromes results in a system that has no polarity (38). That is, a pair of these sites placed on a single piece of DNA recombine to give a mixture of inversions and deletions. Despite this convincing result and its demonstration in at least one other recombination system (79), there are many examples in which polarity is redundantly encoded, i.e., in the overlap region and elsewhere in the recombination locus. In the E. coli xer system, significant differences exist in the recombinase binding sites of the core. These differences are reflected in the requirement for two related but distinct recombinase proteins, one for each binding site, that impart polarity to the recombination locus even when its overlap region is made symmetrical (7). Furthermore, as discussed below, all members of the resolvase invertase family that have been studied appear to require accessory components that lie outside the core; these impart polarity to the system even when the overlap is symmetrical (39). Although not yet tested, the same is also expected to be true for some members of the integrase family that use accessory factors, for example, the lambda excision system.
A simplified view of the functional core of a recombination locus that summarizes the previous section is shown in Fig. 8A. Although this element and the recombinase protomers that bind to it are the basic ingredients for strand exchange, many (but not all) site-specific recombination systems require additional elements for their function. The extra components usually are associated with an enlargement of the recombination locus beyond the borders of the functional core and typically include extra binding sites for the recombinase and /or binding sites for accessory proteins that are unrelated to the recombinase. For example, a functional recombination locus for the Tn3 resolvase (called a res site) comprises not only a 25-bp functional core region with a pair of inversely repeated resolvase binding sites but also two more corelike elements (Fig. 8B). The Hin and Gin specific DNA inversion systems provide even more striking examples of dependence on an accessory protein (39). Here, in addition to a typical core for binding the recombinase (called hix or gix sites), efficient site-specific inversion requires a segment of DNA that binds the E. coli FIS (factor for inversion stimulation) protein. Since the FIS binding segment can stimulate recombination when positioned at variable distances from the core of the locus, it is described as a recombinational enhancer. Lambda integration requires a combination of accessory components. While the recombination locus on the E. coli chromosome (called the attB attachment site) consists merely of a functional core, the recombination locus carried by the phage (attP) involves extra binding sites for the recombinase, Int, interspersed with binding sites for an accessory protein, IHF (integration host factor), that is encoded by the bacterial host (50) (Fig. 8C). The extra Int binding elements, known as arm sites, are remarkable in that their sequence differs from the binding elements found in the core; they are accordingly recognized by a distinct domain of Int. An extreme level of complexity is found in the loci for lambda excision (50). While one partner (the attL attachment site) comprises a core plus extra binding sites for Int and IHF, the other locus (attR) adds to this mixture a requirement for binding sites for FIS and binding sites for a small viral protein, Xis, first identified by mutations that specifically interfere with lambda excision (Fig. 8D).
Biochemical studies have established that the additional components are not catalytic; breakage and reunion at complex loci is always accomplished by recombinase bound to the core region. Instead, the extra elements seem to be devoted to the construction of higher-order structures, often in combination with the core and its bound recombinase. In some cases, e.g., res and attL, a stable complex involving all the elements of a single recombination locus can be isolated and biochemical studies can then provide insight into their organization (35, 42). In the most spectacular example of this strategy, the molecular structure of the complex between the functional core of the res site and its recombinase has recently been determined by X-ray crystallography (94a). However, it seems likely that the most important higher-order structures are those involving not single but pairs of recombination loci, i.e., synaptic structures. At present it is not clear how complexes involving a single recombination locus relate to such synaptic complexes. The structure of the latter is currently speculative, although a variety of indirect assays are providing useful guideposts (32, 39, 43, 73, 94a). These experiments, taken together with the analysis of complexes formed at single recombination loci, have revealed two features involving higher-order structures that may have wide application: architectural elements and supercoiling.
Double-stranded DNA is a relatively stiff molecule. Biophysical studies predict that it should be difficult to bring together segments of DNA separated by less than 300 bp (91). Yet, individual recombination loci or synaptic structures often demand contact between proteins that are bound to specific sequences that are separated by only 50 to 200 bp (Fig. 8). To assist the formation of these structures, many site-specific recombination systems exploit proteins that deform DNA. For example, IHF protein binds to specific targets within the arm regions of attP and bends the DNA to which it binds (50). The induced deformation is thought to help stabilize a higher-order structure that is needed to position protomers of Int recombinase within attP. That the principal role of IHF at the attachment site is to deform DNA is most directly shown by experiments in which the protein is successfully replaced by unrelated elements that also bend DNA (78). Similarly, in the Hin system, deformation of DNA by HU protein apparently assists contacts between a hix site and a nearby recombinational enhancer (37). Although clearly homologous to IHF, HU protein differs from it in lacking sequence specificity of binding. Nevertheless, HU works in the ∼70-bp region between the enhancer and the recombination locus and is required in concentrations insufficient to coat the remainder of the DNA. Its specificity is apparently derived from its participation in building a complex rather than from an inherent attraction for this region. Evidence against specific interactions between HU and the recombination proteins it assists comes from the observation that HMG-1 and HMG-2, eukaryotic proteins that are known to deform DNA but are unrelated to HU, can replace it in Hin-promoted recombination (69). The view that emerges from these and similar studies is of the operation of architectural elements in the construction of higher-order complexes. Nature has apparently devised, and site-specific recombination systems have exploited, proteins whose principal function is to bend DNA and to thereby assist the formation of nucleoprotein arrays. These accessory proteins probably play similar roles for other complex genetic loci, e.g., replication initiators and terminators, sites for partitioning of chromosomes and packaging of bacteriophages, recombinogenic termini of transposons, etc. In some cases, the accessory proteins might play roles in addition to bending DNA, e.g., making specific contacts with the proteins that they help, but their function as architectural elements is the common theme.
Many site-specific recombination systems strongly depend on supercoiling for their efficient operation. Indeed, DNA gyrase, the bacterial enzyme that introduces negative supercoils into DNA, was discovered by its requirement for in vitro lambda integration (27). Supercoiling affects many properties of DNA and therefore could influence recombination in many ways (40). The most convincing evidence suggests that the tendency of supercoiled DNA to fold on itself (Fig. 9A) is most critical. This is because the contortion of the path of the double helix (formally described as the "writhe" of the helix axis [18]) promotes the wrapping of DNA segments. For example, synapsis in the resolvase systems involves interwrapping of two res sites, and synapsis in the hin/gin systems involves mutual interwrapping of two hix/gix sites together with the recombination enhancer (Fig. 9B). The topology of these interwraps has been established and, in every case, is of the kind that is stimulated by negative supercoiling (40). Similarly, topological and biochemical experiments indicate that, in order to be active, the lambda attP site must adopt a configuration that is strongly enhanced by negative supercoiling (74). Thus, supercoiling plays a similar role to that of the accessory components in that it facilitates the assembly of a higher-order structure. As such, the role of supercoiling in site-specific recombination is likely to be echoed at other complex loci like origins of replication, etc. Of course, supercoiling could play more than one role in recombination. One possibility for an additional role invokes the capacity of supercoiling to melt DNA. This could favor recombination if, for example, separation of the strands of the core were an important step. Another plausible hypothesis invokes the capacity of supercoiling to drive movement of broken strands. By definition, the supercoiled state is not the most stable configuration possible for a circular DNA, but the continuous nature of the double-helix backbone prevents relief of the strain. Accordingly, when supercoiled DNA is cleaved, the broken ends tend to move. It is easy to imagine how such movement could assist the transfer of strands between partners prior to their subsequent ligation. Although these ideas seem attractive and have received some experimental support (40), the role of supercoiling in the creation of higher-order structures appears to predominate.
In the case of the bacteriophage P1 cre recombination system, an efficient and precise rearrangement requires only core sequences and a recombinase that binds to them (38). Similarly, a recombination system that operates on plasmids found in the nucleus of Saccharomyces cerevisiae consists only of a functional core and a recombinase called FLP (15). The effectiveness of these simple systems highlights the added complexity of systems with longer recombination loci and accessory components. The E. coli xer system is a particularly striking case because the xer recombinase is used in both a simple and a complex recombination system (7). On the one hand, a heterodimeric recombinase composed of XerC and XerD proteins is sufficient to recombine a simple core-type locus (dif) that is located at the site of termination of replication in the E. coli chromosome. But on the other hand, the XerCD recombinase requires a longer site and accessory proteins to reduce ColE1 dimers to monomers. The functional core of these ColE1 recombination (cer) sites differs from that of dif sites principally by a change in spacing of the overlap region. Indeed, variant cer sites with dif-like spacing lose the requirement for sequences adjacent to the core as well as the requirement for accessory proteins (86). Similarly, a variant of the Gin recombinase has been isolated that functions without the recombinational enhancer and the FIS accessory protein (19a, 45). These examples suggest that the naturally occurring complex systems may be regarded as simple systems that have evolved to have damaged or weakened functional cores and thus to depend on accessory components. The selective advantage of this strategy becomes obvious when one considers the two additional properties that are associated with the complex systems: orientation specificity and irreversibility.
Simple systems, like the bacteriophage P1 cre system and the yeast FLP system, can recombine two loci that are disposed in all three of the configurations shown in Fig. 1. For such systems, synapsis appears be the result of collision between two loci, and to a first approximation, the success of the collision is not dependent on the configuration of the DNA between them. In contrast, some (but not all) complex systems have evolved an orientation specificity such that recombination is highly favored when both recombination loci are in a particular configuration (direct versus inverted) on the same circular element. For these systems, loci oriented with the inappropriate configuration or loci disposed on separate elements are recombined poorly, if at all. From the best-studied cases we have learned that orientation specificity follows from the complex topology that is associated with an interwrapped synaptic structure (39, 41). Loci on a single circle tend naturally to interwrap, especially if the circle is supercoiled (18). In contrast, loci on separate elements have no natural tendency to interwrap and, for every synaptic interwrap, must make a compensatory (and costly) wrap of the opposite handedness. For two loci on the same circle, directly or inversely repeated loci will be productively aligned for recombination, depending on whether the recombination system has a synaptic topology with an odd or even number of interwraps between loci. In the productive configuration, the overlap regions of the two loci will be aligned so that their homology is evident (Fig. 9B). Loci that are disposed with an unfavorable orientation on a circular DNA can also undergo synapsis. However, in this case the two overlap regions are aligned such that recombination would yield products with mismatched base pairs (Fig. 9C). As described above, such products do not accumulate, presumably because they are processed back into parental configuration.
Orientation specificity focuses a recombination system on the particular job it has evolved to accomplish. For example, the Tn3 and Tn1000 res systems (Fig. 3) are used to reduce cointegrates. Their effectiveness would be hindered if these systems could also generate cointegrates by intermolecular recombination between res sites located on separate circular elements, i.e., could run the resolution reaction backwards. The topological barrier to intermolecular recombination negates this possibility. The orientation specificity of the DNA invertases may be even more critical. Figure 10 shows the probable outcome of a recombination between a pair of inversely repeated sites when each member of the pair is on a different sister chromatid. Recombination generates what could be a lethal disruption of the replication fork. Note, however, that sites on different sisters have a head-to-tail orientation with respect to one another. Thus, for DNA invertases like Gin and Hin, they are subject to the orientation prejudice against directly repeated sites and a disaster is averted. One predicts that all systems that operate on inverted sites will need to avoid this potential disaster. The yeast FLP system apparently does so by placing one of the inverted sites near the replication terminus (25); recombination targets for the β recombinase of plasmid pSM19035 may enjoy a similar arrangement (1a). The strategies employed by other inversion systems such as Fim and shufflon have not yet been examined. In this context, it would be of interest to test the evolutionary fitness of the variant Gin recombinase that has lost dependence on accessory components and thereby has lost orientation specificity (19a, 45).
The lambda integration and excision systems show no orientation specificity; their synaptic structures therefore are expected not to have a topology as elaborate as the resolvase or invertase recombinases. However, the lambda systems epitomize a second way that complex loci can produce a biologically useful outcome: ensuring the irreversibility of recombination. Both in vivo and in vitro studies have clearly shown that lambda integration and excision are unidirectional, i.e., the proteins that promote lambda integration cannot by themselves promote lambda excision, and vice versa (93). This feature follows from the lambda recombination systems’ dependence on sequences that lie outside the functional core. For example, it appears that the arm regions that flank the left and right sides of the attP core (Fig. 8C) must be present together (i.e., in cis) to activate the locus. Integration segregates these arm regions to the two prophage sites and thereby inactivates the system (74). Recombination of the prophage sites, i.e., excision of lambda, is not the simple reversal of the integration reaction; it employs a unique set of accessory factors, Xis and FIS, and a different subset of Int and IHF accessory sites than does lambda integration (50). Excision is itself irreversible, in part because Xis protein interferes with the integration pathway. The fact that lambda integration joins the phage and host chromosomes but cannot separate them, while lambda excision does the opposite, clearly assists the efficient attainment of a sensible biological result. During the establishment of lysogeny, the virus becomes committed to a long-term association with the host; accidental excision of an integrated prophage would be wasteful. Conversely, induction of a lysogen commits lambda to extrachromosomal growth; it would be counterproductive to reintegrate an excised viral chromosome. These benefits have presumably provided the selective force for the evolution of much of the complexity of the lambda recombination system. Although other phage systems may use a different set of accessory factors and binding sites, similar considerations probably account for their complexity. Of course, where selective pressure for irreversibility is lacking, as might be the case for movement of transposons or integron cassettes, integration/excision systems are expected to resemble the P1 cre system in complexity.
It has been argued that complex nucleoprotein arrays are needed for fidelity and efficiency of macromolecular transformations (22). Conservative site-specific recombination systems provide little support for this view. The systems that consist only of a functional core are faithful and efficient without the benefit of higher-order structures. And the complex systems have used such assemblages not for improving fidelity and speed but to achieve a novel property: selectivity in the reactivity of substrates and products.
Virtually all site-specific recombination systems exhibit tight control over the positioning of the rearrangements that they promote, i.e., where they occur in the genome. In contrast, there is a bimodal distribution in the degree to which the timing of rearrangements is controlled. At one end of the spectrum lie site-specific recombination systems that use DNA rearrangement as part of a developmental program. Here, as typified by the bacteriophage integration/excision systems, recombination is only turned on at an appropriate period in the life of the organism. At the other extreme of the spectrum lie those systems, typified by the S. typhimurium Hin-promoted inversion, that are essentially constitutive. The key difference between the two ends of the spectrum is the degree to which the synthesis of recombinase is controlled.
The induction of a lysogen is a classic paradigm for a developmental program in prokaryotes (72). Many of the switches that are turned during prophage induction are epigenetic; i.e., they involve changes in gene expression but do not alter the genome. The sole genetic change is the excision of the prophage DNA from the chromosome. Since excision is essential for efficient replication and packaging of the bacteriophage DNA, it must be tightly coupled to the remainder of the developmental program. In the case of phage lambda this coupling is achieved because, in a lysogen, the synthesis of Int and Xis proteins is under the control of the lambda repressor. Thus, these essential components of the lambda excision reaction are only made when lambda repressor is inactivated and the entire lysis program is induced (72). A different control strategy must be applied to properly regulate the creation of an integrated prophage. Here, it is important to achieve synthesis of Int but not Xis and, moreover, to do this in a cell that is accumulating sufficient repressor to turn off the lytic pathway. This is neatly accomplished by a lambda protein, cII, that is made early after infection and activates transcription from several repressor-insensitive promoters, one of which transcribes the int (but not the xis) gene (72). Thus, just as for excision, the efficiency and timing of lambda integration are set by the appropriate synthesis of the viral gene products needed for recombination. In contrast, regulation of the host contribution to lambda integration and excision is less prominent. This is even the case for IHF, which not only is needed as a component of the integration and excision reactions, but also influences the production and/or accumulation of the cII regulator of lysogeny (24). Variations occur in the concentration of IHF under various growth conditions (21), and the degree of supercoiling is thought to vary physiologically (26). However, the range of these variations is small and their effects on recombination frequency should be correspondingly small. Indeed, even though removal of FIS by mutation has clear effects on lambda lysogeny, it has been difficult to demonstrate that the large variation in FIS concentrations that accompanies growth (23) influences the efficiency of lambda excision. Other regulated systems have not been studied in as much detail as those involved in bacteriophage integration and excision. However, in the developmental programs of Anabaena and Bacillus, it appears that synthesis of recombinase is also the control mechanism (33).
A constant, low-level production of recombinants is appropriate for the biological function of systems which serve to create genetic variation in a population of bacteria. For example, the constitutive production of Hin recombinase ensures that a population of Salmonella that starts from a single cell with one kind of flagellum will contain a fraction of cells with the alternate flagellar form. Exposure to phages or antibodies that specifically attack the predominant flagellar form therefore will not annihilate the population. The Hin recombinase is produced at low levels and sponsors a correspondingly low level of inversion (9). It is possible to alter the frequency of Hin-promoted inversion by mutation, e.g., by disrupting the genes for HU or FIS. However, just as in the case of phage lambda, there is little evidence suggesting that natural variation in these components is used to regulate the frequency or timing of recombination. Moreover, aside from a possible autoregulation that serves to keep the synthesis of Hin at a low and constant level (80), there appear to be no major controls over the timing and efficiency of Hin expression; the system is autonomous. A similarly constitutive expression characterizes the gin recombinase of bacteriophage Mu (70). Because they serve to generate population variants, the same is expected for the Fim, Piv, and shufflon recombinases, but few or no studies exist.
The term "programmed rearrangements" has been applied to the reactions promoted by both constitutive and regulated recombinases (8). Since the term "programmed" generally implies a developmental program that is called into play under appropriate conditions, it is somewhat confusing to apply it to the constitutive rearrangements. It might be more suitable to describe the entire group of site-specific recombinations as "targeted" or "discriminate" rearrangements (60), to emphasize that the events are usually focused to specific genetic loci. The term "programmed rearrangements" would then be reserved for those recombination systems that are controlled in time as well as place.
The study of site-specific recombination in E. coli and S. typhimurium has blossomed in the years since Campbell’s hypothesis provided a guidepost for research. The task of understanding these reactions has been greatly aided by their defining criterion: site specificity. Thus, it has been relatively straightforward to understand the biological role of a rearrangement because one can focus on a particular segment of the genome that is being manipulated. In addition, the specific nature of these rearrangements has greatly aided their biochemical analysis by providing landmarks that signal their faithful reconstruction in cell-free systems. Such reconstructions have also been helped by the fact that, in the main, cells have a single predominant mechanism for each reaction so that investigators are not troubled by artificial or alternative in vitro reactions.
The maturing of the site-specific recombination field now opens the way for new challenges to be addressed. For example, the discovery of chromosomal rearrangements in B. subtilis and A. variabilis that are part of developmental programs raises the possibility that E. coli and S. typhimurium also contain recombination-sponsored developmental programs that await discovery. It remains to be seen whether either of the two major families of site-specific recombination systems that are operative in these organisms will be discovered in metazoans and, if so, what roles they might play in the biology of complex organisms. For the bacterial recombination systems whose function is known and whose biochemistry is already well described, a major challenge is to convert biochemical facts into an understanding of enzymological mechanism. Fascinating questions about the pathway by which partner DNAs find each other in a timely fashion, the structure of synaptic and strand cleavage intermediates, and the way that DNA strands move between partners represent the next frontier toward a deeper understanding of these reactions. Such understanding should provide a pleasing insight into the way nature has arranged to manipulate genomes. Moreover, since site-specific recombination underlies critical events in the life of many pathogens (lysogeny in mycobacteria, antigenic variation in salmonellae, acquisition of antibiotic resistance in streptococci, etc.), the study of these reactions will play an important role in a new generation of medical applications. Finally, the complexity of site-specific recombination systems is comparable to that of many other transactions involving DNA. Therefore, the techniques and principles learned from the advanced study of site-specific recombination should continue to benefit our understanding of the workings of promoters, origins of replication, and other specific elements of the genome of E. coli, S. typhimurium, and, by extension, all organisms.
This chapter is designed as a guide for the uninitiated; as such it presents a streamlined and highly simplified view of a complex and exciting field. I apologize to my colleagues whose important work is adumbrated or omitted from discussion. I express my gratitude to Ravi Allada and Reid Johnson for the comments on this chapter and to Brooks Low for encouragement and advice. I also thank Zaida Zanata and Monese Christensen for preparation of the manuscript and Martha Blalock for preparation of the figures.
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