Site-specific recombination Xis-independent excisive recombination of bacteriophage lambda

The integration of bacteriophage lambda into the Escherichia coli chromosome depends on the phage-encoded Int protein; prophage excision depends on Int and a second phage function, Xis. Limited excisive recombination has been observed in vivo with certain xis mutants, suggesting that Int may be able to carry out excision without Xis.We report here that purified Int protein carries out lambda site-specific excisive recombination in vitro in the absence of Xis. This reaction requires host factors derived from a non-lysogenic E. coli strain and is influenced strongly by ionic strength. Excision in the absence of Xis occurs slowly at low salt concentrations (40 mm-NaCl) and very little excision occurs at high salt concentrations (100 mm-NaCl). In the presence of Xis, excisive recombination proceeds rapidly at both low and high ionic strengths. These observations are consistent with previous experiments that suggested the partial dispensability of Xis for excision.     

Site-specific recombination in bacteriophage lambda

Lambda attB-attP is a derivative of bacteriophage lambda that contains both attB and attP, two sites required for integrative recombination. Lambda attB-attP can undergo int-mediated recombination to yield progeny phages whose DNA is 15% shorter than that of the parental phage. We have studied intracellular phage DNA from an infection of lysogenic bacteria with λattB-attP in the presence of int gene product, rifampicin and chloramphenicol. The majority of the intracellular phage DNA consisted of circles with lengths of 17.51, 15.09 and 2.38 μm. Partial denaturation mapping confirmed that the 15.09 and the 2.38-μm molecules arose by an int-mediated intramolecular recombination reaction of the type predicted by the Campbell (1962) model. A minor proportion of the circles (3%) were much larger (33.9, 30.2 and 4.7 μm); in these cases denaturation mapping indicated that both intra- and intermoleeular recombination could take place.     

Dimeric intermediates of recombination in phage lambda

Biparental lambda phage DNA dimers formed by the Rec recombination system of E. coli were isolated in the absence of DNA replication and phage maturation. The RecA but not the RecB gene is required for dimer formation. Dimers are primarily circular but can also be branched circular or linear. In circular dimers the crossover points are distributed uniformly along the chromosome, even in the presence of the RecB-dependent Chi recombinational hotspots. Thus in the absence of DNA synthesis and maturation, the Rec system can act reciprocally both in the presence and absence of the RecB gene; this lack of RecB participation accounts for the observed lack of Chi activity.     

Purification of the bacteriophage lambda xis gene product required for lambda excisive recombination

Excision of the lambda prophage from the chromosome of its Escherichia coli host requires the products of the two viral genes int and xis. This paper reports a purification of the lambda xis gene product using a complementation assay in which functional Xis must be added to purified Int and an E. coli-derived host factor extract. Excisive recombination between a left (attL) and right (attR) prophage attachment site cloned on the same plasmid DNA substrate occurred efficiently under these conditions. Purified Int and Xis together could not carry out excision in vitro unless an extract derived from the E. coli host was added; purified integration host factor satisfied this requirement. Xis appears to have a molecular weight of 8800 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It possesses no detectable endonuclease or topoisomerase activities, does not appear to bind DNA to filters, and does not increase the ability of Int to bind DNA. The addition of Xis not only stimulated excisive recombination in vitro but also inhibited integrative recombination. Xis protected Int protein from heat inactivation, suggesting a possible interaction between the two proteins. In light of these observations, possible roles for Xis in recombination are discussed.     

Generalized recombination in tandem duplications of bacteriophage lambda

Summary Recombination between the tandem duplicated segments of b221a106-15 yields unduplicated (single-copy) b221 phage. The apparent frequency of intramolecular events among these recombinations was determined for both cellular (Rec) and bacteriophage (Red) generalized recombination systems. The progeny from single-cycle growth experiments with genetically marked duplication phages were treated with EDTA to inactivate all but the singlecopy phages produced by recombination. Analysis of the genotypes of the EDTA-resistant phages suggested that intramolecular events were about 1 to 5 times as frequent as intermolecular ones. While the results suggest that intramolecular events are not intrinsically forbidden, the quantitative values for the ratio depend on the assumption that intracellular phage chromosomes are completely mixed.     

Orientation-dependent recombination hotspot activity in bacteriophage lambda.

Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi⁺ phenotype in one orientation and Chi^? in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense.When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.     

Bacteriophage lambda cloning vehicles for studies of genetic recombination

A pair of bacteriophage lambda cloning vehicles has been constructed for use in studies of genetic recombination. These phages, lambda rva and lambda rvb, have the following properties: (1) Each vector has a single HindIII site in the immunity region, at which segments of DNA can be inserted. (2) These HindIII sites are flanked by selectable markers with the following phenotypes: Spi+/- (Fec+/-) to the left, and imm lambda or imm434 to the right. (3) There is essentially no sequence homology between the two phages in this region, so recombination of the markers at reasonable frequency depends on the presence of homologous inserts at the HindIII sites. As a consequence, recovered recombinants must have resulted from a crossover event within the insert DNA. Restriction enzyme maps of the vectors have been determined. Variants of the original vectors have been isolated which permit separate examination of the viral (Red) and bacterial (Rec) generalized recombination mechanisms, and which provide a standard interval to which frequencies of recombination in cloned DNAs can be compared.     

Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate – strand annealing or strand invasion – accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.     

Regulation of site-specific recombination by the C-terminus of lambda integrase

Site-specific recombination catalyzed by bacteriophage λ integrase (Int) is essential for establishment and termination of the viral lysogenic life cycle. Int is the archetype of the tyrosine recombinase family whose members are responsible for DNA rearrangement in prokaryotes, eukaryotes and viruses. The mechanism regulating catalytic activity during recombination is incompletely understood. Studies of tyrosine recombinases bound to their target substrates suggest that the C-termini of the proteins are involved in protein–protein contacts that control the timing of DNA cleavage events during recombination. We investigated an Int truncation mutant (W350) that possesses enhanced topoisomerase activity but greater than 100-fold reduced recombination activity. Alanine scanning mutagenesis of the C-terminus indicates that two mutants, W350A and I353A, cannot perform site-specific recombination although their DNA binding, cleavage and ligation activities are at wild-type levels. Two other mutants, R346A and R348A, are deficient solely in the ability to cleave DNA. To explain these results, we have constructed a homology-threaded model of the Int structure using a Cre crystal structure. We propose that residues R346 and R348 are involved in orientation of the catalytic tyrosine that cleaves DNA, whereas W350 and I353 control and make intermolecular contacts with other Int proteins in the higher order recombination structures known as intasomes. These results suggest that Int and the other tyrosine recombinases have evolved regulatory contacts that coordinate site-specific recombination at the C-terminus.     

Extent of sequence homology required for bacteriophage lambda site-specific recombination

Bacteriophage lambda integration and exicision occur by reciprocal recombination within a 15-base homologous core region present in the recombining attachment (att) sites. Strand exchange within the core occurs at precise nucleotide positions, which define an overlap region in which the products of recombination contain DNA strands derived from different parents. In order to define the role of sequence homology during recombination we have constructed point mutations within the core and assayed their effects in vivo and in vitro on site-specific recombination. Two of the mutations are located at position ?3 of the core, which is one base-pair outside of the overlap region where strand exchange occurs. These mutations do not affect integrative or excisive recombination, thereby suggesting that homology outside the overlap region is not required for recombination. Two other mutations are located at position ?2 of the core, which is one base-pair within the overlap region. These mutations show severely depressed integrative and excisive recombination activities in vitro and in vivo when recombined against wild-type att sites. However, the ?2 mutations show normal recombination activity when recombined against att sites containing the homologous mutation, thereby suggesting that homology-dependent DNA interactions are required within the overlap region for effective recombination. In vitro recombination between homoduplex attP sites and heteroduplex attB sites demonstrated that the DNA interactions require only one strand of the attB overlap region to be homologous to attP in order to promote recombination.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Involement of supertwisted DNA in integrative recombination of bacteriophage lambda.

Negatively supertwisted closed circular DNA is the primary substrate for integrative recombination of phage λ DNA in vitro. Closed circular λ DNA without supertwists must be converted to the supertwisted form by the action of Escherichia coli DNA gyrase before efficient recombination can occur. When negatively supertwisted substrate is provided, E. coli DNA gyrase and its cofactors are dispensable components of recombination reaction mixtures. In the absence of DNA gyrase activity, circular DNA considerably less negatively twisted than naturally occurring supercoils is an effective substrate, but positively supertwisted DNA appears to be an ineffective substrate.The predominant products of integrative recombination in vitro are covalently closed circles. The closure of the recombined sites appears to occur without appreciable DNA synthesis and without the action of E. coli DNA ligase. No detectable difference can be observed between the degree of supertwisting of product DNA and that of unrecombined DNA. These facts suggest that the resealing of broken DNA strands is an integral part of the recombination reaction mechanism and is closely coupled with the breakage and realignment steps of recombination.     

Viewing single lambda site-specific recombination events from start to finish

The site-specific recombination pathway by which the bacteriophage lambda chromosome is excised from its Escherichia coli host chromosome is a tightly regulated, highly directional, multistep reaction that is executed by a series of multiprotein complexes. Until now, it has been difficult to study the individual steps of such reactions in the context of the entire pathway. Using single-molecule light microscopy, we have examined this process from start to finish. Stable bent-DNA complexes containing integrase and the accessory proteins IHF (integration host factor) and Xis form rapidly on attL and attR recombination partners, and synapsis of partner complexes follows rapidly after their formation. Integrase-mediated DNA cleavage before or immediately after synapsis is required to stabilize the synaptic assemblies. Those complexes that synapsed (approximately 50% of the total) yield recombinant product with a remarkable approximately 100% efficiency. The rate-limiting step of excision occurs after synapsis, but closely precedes or is concomitant with the appearance of a stable Holliday junction. Our kinetic analysis shows that directionality of this recombination reaction is conferred by the irreversibility of multiple reaction steps.     

Protein-protein interactions in a higher-order structure direct lambda site-specific recombination

The highly directional site-specific recombination of bacteriophage lambda is tightly regulated by the binding of three different proteins to a complex array of sites. The manner in which these reactions are both stimulated and inhibited by co-operative binding of proteins to specific sites on the P arm of attP and AttR has been elucidated by correlation of nuclease protection with recombination studies of both wild-type and mutant DNAs. In addition to co-operative forces, there is a specific competitive interaction that allows the protein-DNA complex to serve as a "biological switch". This switch does not depend upon the simple occlusion of DNA binding sites by neighboring proteins; but, rather, the outcome of this competition is dependent on long-range interactions that vary between the higher-order structures of attP and attR. These higher-order structures are dependent on cooperative interactions involving three proteins binding to five or more sites.     

Topology of Xer recombination on catenanes produced by lambda integrase.

Xer site-specific recombination at the psi site from plasmid pSC101 displays topological selectivity, such that recombination normally occurs only between directly repeated sites on the same circular DNA molecule. This intramolecular selectivity is important for the biological role of psi, and is imposed by accessory proteins PepA and ArcA acting at accessory DNA sequences adjacent to the core recombination site. Here we show that the selectivity for intramolecular recombination at psi can be bypassed in multiply interlinked catenanes. Xer site-specific recombination occurred relatively efficiently between antiparallel psi sites located on separate rings of right-handed torus catenanes containing six or more nodes. This recombination introduced one additional node into the catenanes. Antiparallel sites on four-noded right-handed catenanes, the normal product of Xer recombination at psi, were not recombined efficiently. Furthermore, parallel psi sites on right-handed torus catenanes were not substrates for Xer recombination. These findings support a model in which psi sites are plectonemically interwrapped, trapping a precise number of supercoils that are converted to four catenation nodes by Xer strand exchange.