Site-Specific Recombination:Integration, Excision, Resolution, and Inversion of Defined

DNA SegmentsHOWARD A. NASH

125INTRODUCTION

Genetic stability is a cornerstone of life. But, against the background of genome stability, nature toleratesand occasionally favors a modest degree of variation. One kind of variation involves specificrearrangements of DNA. Such events are distinguished from other types of recombination in that they arefocused at special sites in the genome. The concept of recombination between specific loci came fromstudies of the integration and excision of bacteriophage lambda. Allan Campbell’s proposal for theseprocesses (12) had enormous success in guiding genetic experiments on the nature of the putativerecombination sites and the genes that direct their reciprocal joining. These experiments (31) solidlyestablished site-specific recombination as a paradigm for variation in the genome. Since the late 1970s, theemergence of techniques for physical examination and manipulation of DNA has led to the discovery ofmany more systems that exploit rearrangement at specific sites. This chapter first presents the features thatthese systems share with each other and the different ways they contribute to the biology of bacteria andaccessory genetic elements such as bacteriophages, plasmids, and transposons. Later sections outline whatis understood about the way specific recombinases promote these events and how accessory proteins assistsome of these recombinases. Finally, the means of controlling the timing and efficiency of theserecombination events will be surveyed and a few speculations on questions for future research will bepresented.

FEATURES COMMON AMONG THE SITE-SPECIFIC RECOMBINATION SYSTEMS OFPROKARYOTES

Specificity and Polarity of Recombination Loci

One can think of many rearrangements of the genome as a genetic exchange between two partners. In atypical site-specific recombination, both partners carry a well-defined specific site that is necessary for therecombination event and that contains the point of genetic exchange. The degree to which these sites arespecified dictates the uniqueness of the rearrangement. For example, in lambda integration the same pointin the 46.5-kbp viral chromosome is involved in virtually all events and, even more impressively, in thevast 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 highlyspecified, and as a result, the rearrangement is uniquely defined. Exceptions to this rule provide aninteresting set of variations that can be related to the main theme. Of course, recombination loci are not theonly well-defined sites in E. coli and Salmonella typhimurium (official designation, Salmonella entericaserovar Typhimurium). For example, origins of replication, sites of viral packaging, promoters, origins ofchromosome mobilization, etc., are also highly specified. What distinguishes the loci of site-specificrecombination is that they function not singly but in pairs. Nevertheless, studies of the defined targets ofsite-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 orsymmetric) site cannot specify relative orientation. In naturally occurring site-specific systems, therecombination locus of both partners is polar. Thus, these systems have not only positional specificity butalso orientational specificity, a feature that is often important for their biological function and one whichraises interesting mechanistic questions.

Specialized Recombinases

What directs the recombination of these specialized sites? A formal possibility is that the sites are simplyregions 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 specificrecombinase, is devoted to the task. For example, in the case of bacteriophage lambda, integration is foundto 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 ofaction, 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 tocontrol the timing and efficiency of the rearrangement that it promotes. Moreover, specific binding of therecombinase to the recombination locus is an important part of the mechanism for selecting the site.

Breakage and Reunion

The final feature that characterizes the site-specific systems is the “conservative” nature of therecombination event. The term “conservative” means that nothing is degraded or added during therecombination, a description that applies at several levels of molecular detail. First, no genetic informationis lost or gained as a consequence of recombination; the sequence of the rearranged segments is simply apermutation of the parental DNA. A second conservative feature of site-specific recombination is theabsence of replication. Not only is the genetic information of the parents retained in the recombinants, butthe actual nucleotides that make up the parental DNA are conserved. Thus, site-specific recombination isthe archetype of break-and-join recombination and has no contribution from copy-choice or break-copymechanisms (94). This conservative feature distinguishes site-specific recombination from transposition, inwhich 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 ofconservation in site-specific recombination is thermodynamic. In every case tested, breakage and reuniontake place without the intervention of high-energy cofactors (such as ATP), implying that the energy storedin 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 recombinationsystems as precise and delicate tools for rearranging the genome, a feature exploited both in nature and,increasingly, by genetic engineers.

BIOLOGICAL ROLES FOR SITE-SPECIFIC RECOMBINATION

Rearrangement of Chromosomes

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 isconvenient to group these systems by the structural consequences of their action. Figure 1 contrasts threepossible ways that two polar sites may be distributed in a genome. Each site can be situated on a separateelement 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 asingle unit. (Recombination between loci on separate linear elements or between a linear and a circularelement 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, resolutionor excision (Fig. 1C), recombination splits one element into two.

Biological Consequences of Rearrangement

How are these different arrangements of recombination loci used to produce biologically meaningfuloutcomes? The best-known examples come from systems in which this kind of recombination was firstdiscovered. 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 geneticinformation (the circular chromosome of bacteriophage lambda). The result is a lysogen, an E. coli bearingan integrated lambda chromosome (prophage).

The two systems that epitomize site-specific inversion (Fig. 2) are used to generate diversity in geneexpression. 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 importantstructural and regulatory genes (Fig. 2A). These two alternative arrangements of the control region neatlyaccount for the phase variation in S. typhimurium, a phenomenon discovered in the 1920s and shown bygenetic experiments in the 1950s to be linked to a “heritable variation” in the control region (52). In asimilar way, inversely oriented recombination sites encompass two open reading frames that encodealternate carboxy termini for a tail fiber gene of bacteriophage Mu (Fig. 2B). Recombination positions oneor the other of these open reading frames adjacent to a common amino-terminal segment and therebyprovides 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 lysogenregenerates a free lambda chromosome and a cured E. coli chromosome (12). Recombination of directlyrepeated 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 acointegrate, in which the two parental circles are joined and a copy of Tn3 is present at each of the two joinpoints. 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 replicationaltransposition, Tn3 also contains a site-specific recombination system (3). Recombination between thespecific locus in each copy of the transposon resolves the cointegrate into two circular elements, each of whichhas a new copy of Tn3 (Fig. 3).

FIGURE 1 Structural consequences of site-specific recombination. Duplex DNA is depicted by a singleline; lowercase letters provide identification and orientation markers along these "chromosomes." Eachrecombination locus is depicted by an arrow (not to scale). Recombination occurs within the locus so as tocreate a hybrid arrow and to rearrange the chromosome. Diagrams illustrate the outcome of recombinationbetween (A) loci on separate chromosomes, (B) loci that are inversely repeated on a single chromosome,and (C) loci that are directly repeated on a single chromosome.

Widespread Distribution of Recombination Systems

These few biological functions—acquisition and elimination of defined segments of DNA, generation ofdiversity in gene expression, and reduction of dimeric forms—appear to be the raison d’être for an impressivearray of different site-specific recombination systems. A survey of such elements is presented in Table 1. Toprovide a sense of the scope of site-specific recombination in the bacterial world, the table is not limited tosystems known to operate in E. coli and S. typhimurium. In the table each recombination system is assigned afunction, but the reader should be aware that in some cases this has not been proven. Moreover, even whenbiological experiments have clearly established that recombination performs the indicated function, it is possiblethat the same recombination components can be used for additional purposes; the listed functions are notexclusive. Even within a given functional grouping, there are important variations. For example, generation ofdiversity in gene expression can either utilize inversely repeated sites, as described in Fig. 2, or directly repeatedsites, as in the removal of segments of DNA that interrupt genes in Bacillus subtilis (85) and Anabaenavariabilis (49). Another variation involves the complexity of DNA that undergoes rearrangement. Forintegration and excision into and out of the bacterial chromosome, the elements range in size from bacteriophagegenomes (lambda), through plasmids (pSAM2) and nonduplicative transposons (Tn916), to single genes

(integrons).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 homologousrecombination between sister chromosomes. At the time of cell division, segregation of these dimers to onedaughter or the other can severely imbalance the orderly inheritance of the affected element. Bacteriophage P1encodes 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 ColE1family exploit an E. coli-encoded site-specific recombination system (xer) to resolve multimeric forms andenhance 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, atevery generation site-specific recombination operates on the entire E. coli genome. Deletion of the dif locus orinactivation 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 thefitness of E. coli.

CORE REGION OF RECOMBINATION SITES AND ENZYMOLOGY OF STRAND TRANSFER

The Functional Core

Any conservative site-specific recombination is likely to involve several mechanistic steps that are at leastconceptually distinct. The DNA of two recombination loci must first be recognized. In a step calledsynapsis, the loci must then be brought into physical contact. After cleavage of the synapsed loci, thebroken ends must then be transferred to new partners and rejoined. For several of the systems describedabove, the recombination pathway has been analyzed at genetic and biochemical levels. Using thispowerful combination of approaches, workers have dissected the recombination loci into their componentparts, identified the gene products responsible for interacting with these parts, and established the principlesby which the resulting protein-DNA assemblies effect a genetic rearrangement. Remarkably, despiteconsiderable diversity in the function, complexity, and evolutionary history of these recombinationsystems, a common theme has emerged. At the heart of each recombination system is a 20- to 30-bpsegment 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 ofthe elementary steps of site-specific recombination involves the DNA of the functional core and itsbound recombinase. First, specific recognition of the core by the recombinase is a major factor in thefidelity with which DNA segments are singled out as targets for rearrangement. Second, pairs of theserecombinase-core DNA complexes interact to form the synaptic structure. Finally, enzymatic activities ofthe bound recombinase protomers catalyze cleavage of core DNA and, after suitable realignment of thebroken ends, ligation to the recombinant configuration. Although all site-specific systems share thistheme, distinctions between different systems emerge from the particular way the elementary steps arecarried out. Because they involve covalent change, the steps involving breakage and reunion of DNAhave been the most completely characterized and are therefore most useful as distinguishing features.

FIGURE 2 Functional consequences of DNA inversion. (A) Phase variation in S. typhimurium. Two genes,H2 and rh1, respectively encode a flagellar subunit and a repressor of an alternate flagellar subunit, H1 (96).When appropriately oriented (top), a promoter (P) can transcribe both genes (indicated by a wiggly line) andthereby ensure exclusive production of the H2 flagellin. When oppositely oriented (bottom), the promotertranscribes neither H2 nor rh1 and thereby permits exclusive expression of the distant flagelling gene H1. Site-specific inversion shuttles the promoter between these two orientations. (B) Host range variation ofbacteriophage Mu. A gene encoding a tail fiber protein, S, contributes to the host range of the phage (29). From apromoter that lies outside the region shown, the phage transcribes one of two alternative forms of the gene, ScSvor ScS'v, which have a common amino terminus but alternative carboxy termini. These forms are constructed byrecombination between loci that are positioned at the junction between the constant (amino) and variable(carboxy) portions of the gene. The recombination loci do not disrupt the reading frame.

FIGURE 3 Resolution of dimeric cointegrates. The mobile element Tn3 is depicted as a rectanglebounded by two filled arrowheads; a site-specific recombination locus (res) within the element is indicatedby an open arrow. Transposition involves fusion of the chromosome carrying Tn3 to the targetchromosome; fusion is coupled with replication of the transposon. In the resulting cointegrate (middlepanel), two copies of the res locus (one per transposon) are oriented in a head-tail fashion. Site-specificrecombination between res loci (3) resolves the cointegrate into two independent chromosomes (bottompanel).

TABLE 1 Site-specific recombination systemsFunction Element or recombinase Host or context Recombinase family ReferenceDiversitygene expression hin S. typhimurium Resolvaseinvertase 39

gin Bacteriophage Mu Resolvase/invertase 41pin E. coli Resolvase/invertase 30SpoIV cisA Bacillus Resolvase/invertase 77xisF Anabena Resolvase/invertase 14fim E. coli Integrase 57Shufflon E. coli plasmid R64 Integrase 47piv Moraxella Unique 54xisA Anabaena Unique 49

Dimer reduction res Transposon Tn3 Resolvase/invertase 82res Transposon Tn1000 Resolvase/invertase 76res Transposon Tn21 Resolvase/invertase 68cre Bacteriophage P1 Integrase 1xer E. coli Integrase 7D protein E. coli plasmid F Integrase 51

Integration/excision Bacteriophage λ E. coli K-12 Integrase 93 HK22 E. coli K-12 Integrase 62 P2 E. coli C Integrase 95 φ21 E. coli K-12 Integrase 11 P22 S. typhimurium Integrase 55, 80a HP1 Haemophilus Integrase 32a, 36 SSV1 Sulfolobus Integrase 61 L5 Mycobacterium Integrase 53 R4 Streptomyces Resolvase/invertase 56aPlasmid pSAM2 Streptomyces Integrase 81 pT181 Bacillus Unique 28Transposon Tn916 Enterococcus Integrase 13, 55b Tn1545 Streptococcus Integrase 71Integron resistance genes aadA Transposon Tn21 Integrase 56 aacA Plasmid pSA Integrase 16, 32b

Strand Cleavage and Strand Transfer

The cleavage step of recombination involves attack by a recombinase polypeptide on a phosphodiesterbond. Recombination requires four such cleavages, i.e., breakage of both strands in the cores of twopartners. 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 residueof the protein and one segment; in the process, the other segment is liberated (Fig. 4B). Cleavage istherefore not by hydrolysis of the phosphodiester, as would be catalyzed by a nuclease (Fig. 4E and F), butby transesterification with the recombinase.

On the basis of the details of strand cleavage, virtually all the site-specific recombination systems ofTable 1 can be grouped into just two families. On the one hand are the recombinases, typified by lambdaintegrase, that use a tyrosine hydroxyl as the attacking nucleophile and that liberate a segment terminatedby 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 thecleavage point of the complementary strand (Fig. 5, left). On the other hand are the recombinases, typifiedby the enzymes of the resolvase and invertase systems, that use a serine hydroxyl as the attackingnucleophile and that liberate a segment terminated by a 3′ OH (34, 46). In these systems cleavage of onestrand is 2 bp to the 3′ side of the cleavage point of the complementary strand (Fig. 5, right). Thepolypeptide sequences of the recombinases within any one family can be aligned, although there can bevery 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.

FIGURE 4 Chemistry of strand transfer. (Left) Cleavage and rejoining of Dna strands by site-specificrecombinases. A specific phosphodiester of a recombination locus (A) is broken by transesterification withan enzyme residue (B). If the segment of DNA that is liberated by cleavage is replaced by a correspondingsegment from a partner DNA (C), another cycle cycle of transesterification accomplishes a covalent strandexchange (D). (Right) Cleavage and rejoining of DNA strands by nuclease plus ligase. A phophodiester (E)is attacked by a water molecule (F). In a typical protocol for genetic engineering, this would be the result ofrestriction nuclease action. To complete the rearrangement, a new segment of DNA (G) is joined to one ofthe original segments (H) by the action of DNA ligase.

FIGURE 5 Polarity of cleavage in two recombinase families. The 5' and 3' ends of each DNA srand aremarked with a circle and arrowhead, respectively. The positions of cleavage on the top and bottom strandsin the resolvaseinvertase family (right) and integrase family (left) are indicated together with the identity ofthe attacking enzyme residue. Although cleavages on both top and bottom strands are both shown in thesame cartoon, they need not occur simultaneously.

To effect a genetic rearrangement, the cleaved strands from two partners must exchange positions withrespect to each other. The details of such strand transfer are not known for any system, but topologicalexperiments (19a, 41, 48, 64, 81a, 84) indicate that ordered movement is the rule and that there are strongcontrols on the kinds of movement that can occur. Ligation of transferred strands is the converse of thecleavage step: the phosphodiester linking the recombinase protein to one DNA segment is attacked by ahydroxyl 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 andcreates a new phosphodiester bond that joins segments of two parental DNA strands (Fig. 4D). Noexogenous source of chemical energy is required for the rejoining. This is unlike the joining of DNAsegments created by nuclease action, in which DNA ligase must use a high-energy cofactor to convert aDNA 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 samepathway 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 canjoin strands from two partners and thereby create a recombinant molecule. What distinguishes thetopoisomerase function of site-specific recombinases is that they are highly efficient at such strandexchange, presumably because they have the capacity to juxtapose partners prior to cleavage and toencourage strand transfer prior to ligation. In contrast, general topoisomerases usually rejoin broken DNAso as to reconstitute the original pattern of connectivity. For these enzymes, the important changes between thebreakage and reunion steps are purely topological; strand exchange appears to be an uncommon, perhapsaccidental, occurrence. In contrast, site-specific recombinases alter both the topology and the connectivity oftheir substrates.

A complete recombination event requires that both strands of each of two cores undergo breakage andsubsequent ligation. One can imagine two extreme ways to couple these events. On the one hand, cleavage oftop and bottom strands of a single core can be concerted, generating a double-strand break (Fig. 6B). In thiscase, 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 initialexchange between partners involves transfer of a single strand. This creates an intermediate form (Fig. 6F), firstproposed by Robin Holliday and accordingly called a Holliday junction, which contains two parental strands andtwo recombinant strands. Recombination is completed, i.e., the Holliday junction is resolved, when theremaining strand from each parent undergoes cleavage, transfer, and ligation (Fig. 6H). Where studied, all themembers of the resolvaseinvertase family generate double-strand breaks and all members of the integrase familygenerate a Holliday junction intermediate.

The Overlap Region and the Role of Homology

Because there is a physical separation between the positions of exchange in the two strands of a recombinationlocus (Fig. 5), the products of recombination will contain a segment in which each strand is derived from adifferent parent (Fig. 6C and H). The segment, called the overlap region, is 6 to 8 bp long for different membersof the integrase family and 2 bp long in all known members of the resolvaseinvertase family. The overlap regionis a part of the recombination locus that is usually identical between partners. (The reader should be aware thatthe region of sequence identity between partners has often been called the core or, more specifically, thehomologous core of a recombination locus. This need not be identical to the functional core, but the two usuallyshare common elements.) Because of the identity of DNA sequence in the overlap region of the parents, thehybrid nature of the recombinant overlap region can typically be detected only by physical means. Although it istherefore normally genetically silent, the overlap region is an important determinant of site specificity through arequirement for sequence matching. For most systems, any alteration in the sequence of the overlap region of arecombination 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 bothtransitions and transversions from the wild-type overlap sequence are permitted if the identical changes areintroduced into the partner locus. Although changes in the length of the overlap region (75) and a fewsubstitutions that apparently change DNA structure of the region (35, 90) are exceptions, the general rule seemsto be that recombinases do not sense the sequence of the overlap region but instead sense the perfection of itsmatch to a partner. In contrast, recombinase binding depends on sequences of the functional core that are locatedlargely outside the overlap region. Here, natural or engineered differences between partners are tolerated (aslong as an adequate binding site is maintained), showing that sequence matching is restricted to the overlapregion. 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 approximationto identity, not the perfect matching required for site-specific recombination.

Biochemical studies have been useful in demonstrating that the block to recombination caused bynonhomology in the overlap region is only partial. The bulk of the evidence indicates that recombinationinitiates normally in these circumstances but cannot proceed to completion. For example, in an invertasesystem, when a circular DNA with a pair of nonhomologous recombination loci is treated with its cognaterecombination enzyme, no recombinants accumulate, but the substrate shows topological changes (59). Thedetails of these changes indicate that double-strand breaks have been made but that, instead of strandsmoving neatly to their recombinant configuration, more extensive strand motion is followed by rejoining ofthe broken recombination loci to reconstitute the parental connectivity. Similarly, in the lambda integrasefamily, partners with heterology in the overlap region form Holliday junctions but fail to resolve them so asto generate recombinant products (44, 65).

How do the recombinases sense that heterologous partners are attempting to recombine? It seemsattractive to imagine that hybrid overlap regions are actually produced under these circumstances and thatthe 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 ifnonhomologous loci not only suffer double-strand breaks but exchange strands and recombine. Although ithas not been directly tested, these mismatched recombinants could be particularly good substrates for a newround of breakage, strand movement, and reunion; such a second cycle of strand exchange wouldreestablish parental connections (59, 83). For members of the lambda integrase family, movement of theHolliday 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, canmove 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, adistortion 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 junctionsformed from nonhomologous partners (1b, 20, 64a). However, experiments with a variety of other artificialrecombination substrates (9a, 17, 63) provide strong suppport for the traditional view and promptalternative 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 ofrecombining partners (7, 13, 16, 56, 89). These cases are all members of the integrase family but have notyet been analyzed at the biochemical level; it will be of interest to see where the differences lie. Onepossibility concerns the stability of the Holliday junction. In systems that depend strongly on overlaphomology, the Holliday junction is an unstable intermediate that either generates recombinants or is largelyprocessed back to the parental configuration. For the systems that generate recombinants in vivo in theabsence of identical overlap regions, the Holliday junction may be a stable product (58).

FIGURE 6 Concerted versus sequential strand exchange. The DNA strands of two recombination loci arepictured by thin and thick lines; strand polarity is indicated as in Fig. 5. (Left) Concerted cleavage theexchange. Both the top and bottom strands of each locus are attacked (A), generating double-strand breaks(B). Recombinants (C) are produced by rejoining the resulting half sites. (Right) Sequential exchanges.One pair of strands is attacked (D), generating enzyme-linked nicks in two strands of identical polarity (E).These strands are transferred and rejoined to create a Holiday intermediate (F). Melting of base pairs andreannealing of strands to different partners can reposition the point of connection between the parentalduplexes (G), a process described as branch migration (66, 88). Recombinants are produced when thebranch position permits the remaining pair of strands to be exchanged, resolving the Holliday junction (H).

Polarity in Recombination

In general, naturally occurring overlap sequences are nonpalindromic. The asymmetric nature of thissegment of the recombination locus stands in contrast to the remainder of the functional core, whichcomprises nearly or exactly inverted repeats for recombinase binding. The overlap segment is therefore anattractive candidate for the element that provides polarity to the recombination locus. Indeed, changing theoverlap 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 placedon a single piece of DNA recombine to give a mixture of inversions and deletions. Despite this convincingresult and its demonstration in at least one other recombination system (79), there are many examples inwhich 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. Thesedifferences are reflected in the requirement for two related but distinct recombinase proteins, one for eachbinding site, that impart polarity to the recombination locus even when its overlap region is madesymmetrical (7). Furthermore, as discussed below, all members of the resolvase invertase family that havebeen studied appear to require accessory components that lie outside the core; these impart polarity to thesystem even when the overlap is symmetrical (39). Although not yet tested, the same is also expected to betrue for some members of the integrase family that use accessory factors, for example, the lambda excisionsystem.

FIGURE 7 Homologous versus heterologous overlap regions. Each double line represents the 6-bpoverlap region of a hypothetical recombination system. Thin and thick lines are used to delineate theindividual strands of the two parental loci. (Left) Recombination between lock with homologous overlapregions produces re-combinants with perfect Watson-Crick complementarity. (Right) Recombinationbetween loci with nonhomologous overlap regions generates mismatches. These are likely to be unstable asintermediates or products and are expected to be cleaved again and processed back to the parentalconfiguration. It is also possible that strand switching between partners occurs prior to ligation. In that case,the mismatches produced when nonhomologous partners interact should interface with the ligation step andthe strands might simply return to parental configuration (reviewed in reference 9a).

ACCESSORY COMPONENTS FOR SITE-SPECIFIC RECOMBINATION

Elements Outside the Functional Core

A simplified view of the functional core of a recombination locus that summarizes the previous section isshown in Fig. 8A. Although this element and the recombinase protomers that bind to it are the basicingredients for strand exchange, many (but not all) site-specific recombination systems require additionalelements for their function. The extra components usually are associated with an enlargement of therecombination locus beyond the borders of the functional core and typically include extra binding sites forthe recombinase and /or binding sites for accessory proteins that are unrelated to the recombinase. Forexample, 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 morecorelike elements (Fig. 8B). The Hin and Gin specific DNA inversion systems provide even more strikingexamples of dependence on an accessory protein (39). Here, in addition to a typical core for binding therecombinase (called hix or gix sites), efficient site-specific inversion requires a segment of DNA that bindsthe E. coli FIS (factor for inversion stimulation) protein. Since the FIS binding segment can stimulaterecombination when positioned at variable distances from the core of the locus, it is described as arecombinational enhancer. Lambda integration requires a combination of accessory components. While therecombination locus on the E. coli chromosome (called the attB attachment site) consists merely of afunctional core, the recombination locus carried by the phage (attP) involves extra binding sites for therecombinase, Int, interspersed with binding sites for an accessory protein, IHF (integration host factor), thatis encoded by the bacterial host (50) (Fig. 8C). The extra Int binding elements, known as arm sites, areremarkable in that their sequence differs from the binding elements found in the core; they are accordinglyrecognized by a distinct domain of Int. An extreme level of complexity is found in the loci for lambdaexcision (50). While one partner (the attL attachment site) comprises a core plus extra binding sites for Intand IHF, the other locus (attR) adds to this mixture a requirement for binding sites for FIS and binding sitesfor 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 andreunion at complex loci is always accomplished by recombinase bound to the core region. Instead, the extraelements seem to be devoted to the construction of higher-order structures, often in combination with thecore and its bound recombinase. In some cases, e.g., res and attL, a stable complex involving all theelements of a single recombination locus can be isolated and biochemical studies can then provide insightinto their organization (35, 42). In the most spectacular example of this strategy, the molecular structureof the complex between the functional core of the res site and its recombinase has recently beendetermined 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. Atpresent it is not clear how complexes involving a single recombination locus relate to such synapticcomplexes. The structure of the latter is currently speculative, although a variety of indirect assays areproviding useful guideposts (32, 39, 43, 73, 94a). These experiments, taken together with the analysis ofcomplexes formed at single recombination loci, have revealed two features involving higher-orderstructures that may have wide application: architectural elements and supercoiling.

DNA Bending and Writhing in Higher-Order Structures

Double-stranded DNA is a relatively stiff molecule. Biophysical studies predict that it should be difficult tobring together segments of DNA separated by less than 300 bp (91). Yet, individual recombination loci orsynaptic structures often demand contact between proteins that are bound to specific sequences that areseparated by only 50 to 200 bp (Fig. 8). To assist the formation of these structures, many site-specificrecombination 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 deformationis thought to help stabilize a higher-order structure that is needed to position protomers of Int recombinasewithin attP. That the principal role of IHF at the attachment site is to deform DNA is most directly shownby 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 betweena hix site and a nearby recombinational enhancer (37). Although clearly homologous to IHF, HU proteindiffers from it in lacking sequence specificity of binding. Nevertheless, HU works in the ∼70-bp regionbetween the enhancer and the recombination locus and is required in concentrations insufficient to coat theremainder of the DNA. Its specificity is apparently derived from its participation in building a complexrather than from an inherent attraction for this region. Evidence against specific interactions between HUand the recombination proteins it assists comes from the observation that HMG-1 and HMG-2, eukaryoticproteins that are known to deform DNA but are unrelated to HU, can replace it in Hin-promotedrecombination (69). The view that emerges from these and similar studies is of the operation ofarchitectural elements in the construction of higher-order complexes. Nature has apparently devised, andsite-specific recombination systems have exploited, proteins whose principal function is to bend DNA andto thereby assist the formation of nucleoprotein arrays. These accessory proteins probably play similar rolesfor other complex genetic loci, e.g., replication initiators and terminators, sites for partitioning ofchromosomes 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 theproteins 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 discoveredby its requirement for in vitro lambda integration (27). Supercoiling affects many properties of DNA andtherefore could influence recombination in many ways (40). The most convincing evidence suggests thatthe tendency of supercoiled DNA to fold on itself (Fig. 9A) is most critical. This is because the contortionof the path of the double helix (formally described as the “writhe” of the helix axis [18]) promotes thewrapping of DNA segments. For example, synapsis in the resolvase systems involves interwrapping of twores sites, and synapsis in the hin/gin systems involves mutual interwrapping of two hix/gix sites togetherwith the recombination enhancer (Fig. 9B). The topology of these interwraps has been established and, inevery case, is of the kind that is stimulated by negative supercoiling (40). Similarly, topological andbiochemical experiments indicate that, in order to be active, the lambda attP site must adopt a configurationthat is strongly enhanced by negative supercoiling (74). Thus, supercoiling plays a similar role to that of theaccessory components in that it facilitates the assembly of a higher-order structure. As such, the role ofsupercoiling in site-specific recombination is likely to be echoed at other complex loci like origins ofreplication, etc. Of course, supercoiling could play more than one role in recombination. One possibility foran additional role invokes the capacity of supercoiling to melt DNA. This could favor recombination if, forexample, separation of the strands of the core were an important step. Another plausible hypothesis invokesthe capacity of supercoiling to drive movement of broken strands. By definition, the supercoiled state is notthe most stable configuration possible for a circular DNA, but the continuous nature of the double-helixbackbone prevents relief of the strain. Accordingly, when supercoiled DNA is cleaved, the broken endstend to move. It is easy to imagine how such movement could assist the transfer of strands betweenpartners prior to their subsequent ligation. Although these ideas seem attractive and have received someexperimental support (40), the role of supercoiling in the creation of higher-order structures appears topredominate.

Higher-Order Structure and Selective Action of Recombinases

In the case of the bacteriophage P1 cre recombination system, an efficient and precise rearrangementrequires only core sequences and a recombinase that binds to them (38). Similarly, a recombination systemthat 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 addedcomplexity of systems with longer recombination loci and accessory components. The E. coli xer system isa particularly striking case because the xer recombinase is used in both a simple and a complexrecombination system (7). On the one hand, a heterodimeric recombinase composed of XerC and XerDproteins is sufficient to recombine a simple core-type locus (dif) that is located at the site of termination ofreplication in the E. coli chromosome. But on the other hand, the XerCD recombinase requires a longer siteand accessory proteins to reduce ColE1 dimers to monomers. The functional core of these ColE1recombination (cer) sites differs from that of dif sites principally by a change in spacing of the overlapregion. Indeed, variant cer sites with dif-like spacing lose the requirement for sequences adjacent to thecore as well as the requirement for accessory proteins (86). Similarly, a variant of the Gin recombinase hasbeen 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 systemsthat have evolved to have damaged or weakened functional cores and thus to depend on accessorycomponents. The selective advantage of this strategy becomes obvious when one considers the twoadditional properties that are associated with the complex systems: orientation specificity andirreversibility.

FIGURE 8 Simple and complex recombination loci. (A) A simple recombination locus that consists onlyof a 30-bp functional core. Binding sites for a recombinase are shown by arrows, and the overlap region isshaded. The remaining panels use the same scale and symbols. (B) The res locus. In addition to a functionalcore (subsite I) that binds two protomers of the resolvase protein, the locus contains two other corelikeelements (subsites II and III) that also bind recombinase. Strand exchange takes place only at subsite I,presumably because the inter-resolvase spacing of subsites II and III is inappropriate (35). (C) Thebacteriophage lambda attP site. Essential binding sites for the arm domain of Int recombinase are indicatedby wiggly arrows, and binding sites for IHF protein are represented by ovals with the letter H. (D) Thebacteriophage lambda attR prophage site. Note that the arm of attR and the left arm of attP, althoughderived from the same segment of DNA. use a different array of accessory dinbind sites. This arrayincludes sites for FIS (F) and Xis (X) proteins. Although attP contains the Int arm binding site as well asthe Xis and FIS binding sites that are used in attR, these are not occupied during lambda integration.Indeed, their occupancy inhibits integration; conversely, occupancy in attR of the binding sites for Int andIHF that are found in at the extreme left and of attP is deleterious for recombination of attR with the otherprophage site, attL (50).

FIGURE 9 Productive and nonproductive alignment of synapsed recombination loci (adapted fromreference 39). (A) A circular substrate for site-specific inversion. The double helix is represented as asingle line, two inversely repeated recombination loci are shown as filled arrows, and the recombinationenhancer is shown as an open rectangle. Negative supercoiling has induced writhing of the axis of thedouble helix. (B) Synaptic topology of this substrate, induced by the Hin/Gin recombinase, is pictured as anisoform of the writhed substrate. Here, the recombination loci and the enhancer are mutually interwrapped.In this productive alignment, the two overlap regions (one for each locus) are oriented such that breakage,limited movement, and reunion can produce recombinants with perfectly matched overlap regions. (C)When a substrate with directly repeated sites adopts the same synaptic topology, the overlap regions arealigned in an unproductive fashion. Recombinants produced from this alignment would have mismatchedoverlap regions and would be processed by further cycles of breakage, strand movement, and strand joiningto regenerate the parental overlap regions. (D) A substrate with directly repeated sites can only form asynapse with both the correct topology and correct overlap alignment by undergoing additional contortions,which severely limits the effectiveness of this pathway.

Simple systems, like the bacteriophage P1 cre system and the yeast FLP system, can recombine twoloci that are disposed in all three of the configurations shown in Fig. 1. For such systems, synapsis appearsbe the result of collision between two loci, and to a first approximation, the success of the collision is notdependent on the configuration of the DNA between them. In contrast, some (but not all) complex systemshave evolved an orientation specificity such that recombination is highly favored when both recombinationloci are in a particular configuration (direct versus inverted) on the same circular element. For thesesystems, loci oriented with the inappropriate configuration or loci disposed on separate elements arerecombined poorly, if at all. From the best-studied cases we have learned that orientation specificityfollows from the complex topology that is associated with an interwrapped synaptic structure (39, 41). Locion a single circle tend naturally to interwrap, especially if the circle is supercoiled (18). In contrast, loci onseparate elements have no natural tendency to interwrap and, for every synaptic interwrap, must make acompensatory (and costly) wrap of the opposite handedness. For two loci on the same circle, directly orinversely repeated loci will be productively aligned for recombination, depending on whether therecombination 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 isevident (Fig. 9B). Loci that are disposed with an unfavorable orientation on a circular DNA can alsoundergo synapsis. However, in this case the two overlap regions are aligned such that recombination wouldyield 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 toaccomplish. For example, the Tn3 and Tn1000 res systems (Fig. 3) are used to reduce cointegrates. Theireffectiveness would be hindered if these systems could also generate cointegrates by intermolecularrecombination between res sites located on separate circular elements, i.e., could run the resolution reactionbackwards. The topological barrier to intermolecular recombination negates this possibility. The orientationspecificity of the DNA invertases may be even more critical. Figure 10 shows the probable outcome of arecombination between a pair of inversely repeated sites when each member of the pair is on a differentsister 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, forDNA invertases like Gin and Hin, they are subject to the orientation prejudice against directly repeatedsites and a disaster is averted. One predicts that all systems that operate on inverted sites will need to avoidthis potential disaster. The yeast FLP system apparently does so by placing one of the inverted sites nearthe replication terminus (25); recombination targets for the β recombinase of plasmid pSM19035 mayenjoy a similar arrangement (1a). The strategies employed by other inversion systems such as Fim andshufflon have not yet been examined. In this context, it would be of interest to test the evolutionary fitnessof the variant Gin recombinase that has lost dependence on accessory components and thereby has lostorientation specificity (19a, 45).

The lambda integration and excision systems show no orientation specificity; their synaptic structurestherefore 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 usefuloutcome: ensuring the irreversibility of recombination. Both in vivo and in vitro studies have clearly shownthat lambda integration and excision are unidirectional, i.e., the proteins that promote lambda integrationcannot by themselves promote lambda excision, and vice versa (93). This feature follows from the lambdarecombination systems’ dependence on sequences that lie outside the functional core. For example, itappears that the arm regions that flank the left and right sides of the attP core (Fig. 8C) must be presenttogether (i.e., in cis) to activate the locus. Integration segregates these arm regions to the two prophage sitesand thereby inactivates the system (74). Recombination of the prophage sites, i.e., excision of lambda, isnot 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 itselfirreversible, in part because Xis protein interferes with the integration pathway. The fact that lambdaintegration joins the phage and host chromosomes but cannot separate them, while lambda excision doesthe opposite, clearly assists the efficient attainment of a sensible biological result. During the establishmentof lysogeny, the virus becomes committed to a long-term association with the host; accidental excision ofan integrated prophage would be wasteful. Conversely, induction of a lysogen commits lambda toextrachromosomal 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 ofthe lambda recombination system. Although other phage systems may use a different set of accessoryfactors and binding sites, similar considerations probably account for their complexity. Of course, whereselective pressure for irreversibility is lacking, as might be the case for movement of transposons orintegron 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 ofmacromolecular transformations (22). Conservative site-specific recombination systems provide littlesupport for this view. The systems that consist only of a functional core are faithful and efficient withoutthe 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 andproducts.

CONTROL OF THE EFFICIENCY AND TIMING OF SITE-SPECIFIC RECOMBINATION

Virtually all site-specific recombination systems exhibit tight control over the positioning of therearrangements that they promote, i.e., where they occur in the genome. In contrast, there is a bimodaldistribution in the degree to which the timing of rearrangements is controlled. At one end of the spectrumlie 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 anappropriate 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 differencebetween the two ends of the spectrum is the degree to which the synthesis of recombinase is controlled.

FIGURE 10 Recombination between sister chromatids. (A) A typical pair of loci destined for site-specific inversion reside on a chromosome. During replication of this chromosome (B), these loci areduplicated on sisters that have not yet separated. (The figure is not to scale; the origin of replication couldbe far to the left or right from these loci.) Recombination between loci distributed diagonally on the twosisters destroys both chromosomes. For example, panel C shows the result of genetic exchange between theloci at top left and bottom right of panel B. The demonstration that this disastrous outcome is the result ofordinary synapsis and strand transfer between these loci is left as an exercise for the reader.

Regulated Recombinases

The induction of a lysogen is a classic paradigm for a developmental program in prokaryotes (72). Many ofthe switches that are turned during prophage induction are epigenetic; i.e., they involve changes in geneexpression but do not alter the genome. The sole genetic change is the excision of the prophage DNA fromthe chromosome. Since excision is essential for efficient replication and packaging of the bacteriophageDNA, it must be tightly coupled to the remainder of the developmental program. In the case of phagelambda this coupling is achieved because, in a lysogen, the synthesis of Int and Xis proteins is under thecontrol of the lambda repressor. Thus, these essential components of the lambda excision reaction are onlymade when lambda repressor is inactivated and the entire lysis program is induced (72). A different controlstrategy must be applied to properly regulate the creation of an integrated prophage. Here, it is important toachieve synthesis of Int but not Xis and, moreover, to do this in a cell that is accumulating sufficientrepressor to turn off the lytic pathway. This is neatly accomplished by a lambda protein, cII, that is madeearly after infection and activates transcription from several repressor-insensitive promoters, one of whichtranscribes the int (but not the xis) gene (72). Thus, just as for excision, the efficiency and timing of lambdaintegration are set by the appropriate synthesis of the viral gene products needed for recombination. Incontrast, regulation of the host contribution to lambda integration and excision is less prominent. This iseven 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). Variationsoccur in the concentration of IHF under various growth conditions (21), and the degree of supercoiling isthought to vary physiologically (26). However, the range of these variations is small and their effects onrecombination frequency should be correspondingly small. Indeed, even though removal of FIS bymutation has clear effects on lambda lysogeny, it has been difficult to demonstrate that the large variationin FIS concentrations that accompanies growth (23) influences the efficiency of lambda excision. Otherregulated systems have not been studied in as much detail as those involved in bacteriophage integrationand excision. However, in the developmental programs of Anabaena and Bacillus, it appears that synthesisof recombinase is also the control mechanism (33).

Constitutive Recombinases

A constant, low-level production of recombinants is appropriate for the biological function of systemswhich serve to create genetic variation in a population of bacteria. For example, the constitutive productionof Hin recombinase ensures that a population of Salmonella that starts from a single cell with one kind offlagellum will contain a fraction of cells with the alternate flagellar form. Exposure to phages or antibodiesthat specifically attack the predominant flagellar form therefore will not annihilate the population. The Hinrecombinase is produced at low levels and sponsors a correspondingly low level of inversion (9). It ispossible to alter the frequency of Hin-promoted inversion by mutation, e.g., by disrupting the genes for HUor FIS. However, just as in the case of phage lambda, there is little evidence suggesting that naturalvariation 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 isautonomous. 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, andshufflon recombinases, but few or no studies exist.

Programmed Rearrangements

The term “programmed rearrangements” has been applied to the reactions promoted by both constitutiveand regulated recombinases (8). Since the term “programmed” generally implies a developmental programthat 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 tospecific genetic loci. The term “programmed rearrangements” would then be reserved for thoserecombination systems that are controlled in time as well as place.

PERSPECTIVES

The study of site-specific recombination in E. coli and S. typhimurium has blossomed in the years sinceCampbell’s hypothesis provided a guidepost for research. The task of understanding these reactions hasbeen greatly aided by their defining criterion: site specificity. Thus, it has been relatively straightforward tounderstand the biological role of a rearrangement because one can focus on a particular segment of thegenome that is being manipulated. In addition, the specific nature of these rearrangements has greatly aidedtheir biochemical analysis by providing landmarks that signal their faithful reconstruction in cell-freesystems. Such reconstructions have also been helped by the fact that, in the main, cells have a singlepredominant mechanism for each reaction so that investigators are not troubled by artificial or alternative invitro reactions.

The maturing of the site-specific recombination field now opens the way for new challenges to beaddressed. For example, the discovery of chromosomal rearrangements in B. subtilis and A. variabilis thatare part of developmental programs raises the possibility that E. coli and S. typhimurium also containrecombination-sponsored developmental programs that await discovery. It remains to be seen whethereither of the two major families of site-specific recombination systems that are operative in these organismswill 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 welldescribed, a major challenge is to convert biochemical facts into an understanding of enzymologicalmechanism. Fascinating questions about the pathway by which partner DNAs find each other in a timelyfashion, the structure of synaptic and strand cleavage intermediates, and the way that DNA strands movebetween partners represent the next frontier toward a deeper understanding of these reactions. Suchunderstanding 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 instreptococci, etc.), the study of these reactions will play an important role in a new generation of medicalapplications. Finally, the complexity of site-specific recombination systems is comparable to that of manyother transactions involving DNA. Therefore, the techniques and principles learned from the advancedstudy of site-specific recombination should continue to benefit our understanding of the workings ofpromoters, origins of replication, and other specific elements of the genome of E. coli, S. typhimurium,and, by extension, all organisms.

ACKNOWLEDGMENTS

This chapter is designed as a guide for the uninitiated; as such it presents a streamlined and highlysimplified view of a complex and exciting field. I apologize to my colleagues whose important work isadumbrated or omitted from discussion. I express my gratitude to Ravi Allada and Reid Johnson for thecomments on this chapter and to Brooks Low for encouragement and advice. I also thank Zaida Zanata andMonese Christensen for preparation of the manuscript and Martha Blalock for preparation of the figures.

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