Dynamic, structural, and regulatory aspects of lambda site-specific recombination.
Explore the source record for details and available documents.
Bacteriophage lambda↗
Biomedical subjects
A Landy
Publications and source records attributed to A Landy.
Empirical estimation of protein-induced DNA bending angles: applications to lambda site-specific recombination complexes.
Protein-induced DNA bending is an important element in the structure of many protein-DNA complexes, including those involved in replication, transcription, and recombination. To understand these structures, the path followed by the DNA in each complex must be established. We have generated an empirical relation between the degree of bending and the altered electrophoretic mobility in polyacrylamide gels that allows estimation of protein-induced bends. This technique has been used to analyze 17 different protein-DNA complexes formed by six proteins including the four proteins involved in lambda site-specific recombination. The simplicity of this technique should make it useful in estimating angles for the construction of models of protein-DNA complexes and readily applicable to many systems where questions of higher-order structure are important for understanding function.
Bacteriophage lambda↗
Autonomous DNA binding domains of lambda integrase recognize two different sequence families.
The 40 kd lambda Integrase protein is shown to contain two autonomous DNA binding domains with different sequence specificities. Competition experiments in which the binding activity of Int is assayed through nuclease protection demonstrate the functional independence of the two DNA recognition specificities. Proteolytic cleavage of Int and footprinting analysis of the resulting two major peptides allow the physical separation and identification of two DNA binding domains: an amino-terminal peptide that interacts with "arm-type" sites and a carboxy-terminal peptide that binds to "core-type" sequences. In addition, the data suggest that the two domains can bind DNA simultaneously, consistent with a model in which Integrase would link two disparate DNA sequences.
Amino Acid Sequence↗
Suicide recombination substrates yield covalent lambda integrase-DNA complexes and lead to identification of the active site tyrosine.
High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.
Amino Acid Sequence↗
Helical-repeat dependence of integrative recombination of bacteriophage lambda: role of the P1 and H1 protein binding sites.
The efficiency of site-specific recombination of bacteriophage lambda was found to depend on the spacing between distant protein binding sites. Insertions and deletions of up to 30 base pairs were made in the nonessential regions between the H1 and H2 protein binding sites. Recombination was found to occur in substrates with changes of integral multiples of a DNA helical repeat, whereas recombination was defective in substrates with nonintegral changes. The lambda recombinogenic complex is especially interesting because two different proteins are involved: integration host factor (IHF), which has been shown to bend DNA, and the phage-encoded integrase protein (Int), which has been shown to have two distinct DNA-binding domains. The importance of angular displacement of protein binding sites was confirmed by addition of ethidium bromide to defective substrates. Significant stimulation of recombination was observed when sufficient drug intercalated and unwound the DNA to allow improved orientation of sites. The orientation effects are dependent on supercoiling, as spacing is less important in conditions where supercoiling and the P1-H1 sites are not required for recombination.
Bacteriophage lambda↗
Cellular factors couple recombination with growth phase: characterization of a new component in the lambda site-specific recombination pathway.
Here we characterize FIS (factor for inversion stimulation), a new cellular component of the lambda site-specific recombination pathway. This host protein binds to a specific region in the lambda attP overlapping the Xis binding sites and can bind cooperatively with Xis to these sites. FIS stimulates lambda excision up to 20-fold in vitro in the presence of suboptimal Xis concentrations, but has no effect in the presence of saturating Xis; FIS has no effect on integrative recombination. FIS can replace one Xis molecule in a series of cooperative and competitive interactions but cannot carry out excision in the absence of Xis. FIS's role in the regulation of recombination has been inferred from in vivo modification of DNA. In exponentially growing cells the lambda FIS site is fully occupied, whereas in stationary-phase cells this binding site is vacant.
Bacteriophage lambda↗
Site-specific recombination intermediates trapped with suicide substrates.
A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.
Bacteriophage lambda↗
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.
Allosteric Site↗
Structural and regulatory divergence among site-specific recombination genes of lambdoid phage.
The lambdoid bacteriophage phi 80 and P22 have site-specific recombination systems similar to that of lambda. Each of the three phage has a different insertion specificity, but structural analysis of their attachment sites suggests that the three recombination pathways share similar features. In this study, we have identified and sequenced the int and xis genes of phi 80 and P22. phi 80 int and xis were identified using a plasmid recombination assay in vivo, and the P22 genes were mapped using Tn1 insertion mutations. In all three phage, the site-specific recombination genes are located directly adjacent to the phage attachment site. Interestingly, the transcriptional orientation of the phi 80 int gene is opposite to that of lambda and P22 int, resulting in convergent transcription of phi 80 int and xis. Because of its transcriptional orientation, phi 80 int cannot be expressed by the major leftward promoter, PL, and the regulatory strategy of phi 80 integration and excision must differ significantly from that of lambda. The deduced amino acid sequences of the recombination proteins of the three systems show surprisingly little homology. Sequences homologous to the lambda PI promoter are more conserved than the protein-coding sequences. Nevertheless, the Int proteins are locally related in the C-terminal sequences, particularly for a stretch of some 25 amino acid residues that lie approximately 50 residues from the C terminus. The Xis proteins can be aligned at their N termini.
Amino Acid Sequence↗
The integrase family of site-specific recombinases: regional similarities and global diversity.
A combination of two methods for detecting distant relationships in protein primary sequences was used to compare the site-specific recombination proteins encoded by bacteriophage lambda, phi 80, P22, P2, 186, P4 and P1. This group of proteins exhibits an unexpectedly large diversity of sequences. Despite this diversity, all of the recombinases can be aligned in their C-terminal halves. A 40-residue region near the C terminus is particularly well conserved in all the proteins and is homologous to a region near the C terminus of the yeast 2 mu plasmid Flp protein. This family of recombinases does not appear to be related to any other site-specific recombinases. Three positions are perfectly conserved within this family: histidine, arginine and tyrosine are found at respective alignment positions 396, 399 and 433 within the well-conserved C-terminal region. We speculate that these residues contribute to the active site of this family of recombinases, and suggest that tyrosine-433 forms a transient covalent linkage to DNA during strand cleavage and rejoining.
Amino Acid Sequence↗
Mutations in an integration host factor-binding site: effect on lambda site-specific recombination and regulatory implications.
The manner in which integration host factor (IHF) regulates lambda site-specific recombination has been analyzed by examining the behavior of both wild-type and mutant DNAs in integrative and excisive recombination as well as in protein binding. While integrative recombination of an attP with two base changes in the H1 site required 8-fold more IHF than did wild type, binding to this site was lowered at least 500-fold, suggestive of cooperative interactions. A mutant attP with nine base changes did not integrate at all in vitro, with the defect being less severe in vivo. IHF inhibition of excisive recombination was relieved by both mutations in vitro and in vivo. These results imply that occupancy of the H1 site is critical for determining the direction of recombination. It is proposed that IHF inhibition of excision provides a monitor of the strength of the induction stimulus and the nutritional state of the cell; this would allow the prophage to excise selectively in conditions which favor successful completion of the lytic cycle.
Bacterial Proteins↗
Control of directionality in lambda site specific recombination.
The simple relation between the substrates and products of site-specific recombination raises questions about the control of directionality often observed in this class of DNA transactions. For bacteriophage lambda, viral integration and excision proceed by discrete pathways, and DNA substrates with the intrinsic property of recombining in only one direction can be constructed. These pathways display an asymmetric reliance on a complex array of protein binding sites, and they respond differently to changes in the concentrations of the relevant proteins. The Escherichia coli protein integration host factor (IHF) differentially affects integrative and excisive recombination, thereby influencing directionality. A four- to eightfold increase in intracellular IHF coincides with the transition from exponential to stationary phase; this provides a mechanism for growth phase-dependent regulation of recombination that makes the cellular physiology an intrinsic part of the recombination reaction.
Bacterial Proteins↗
The phi 80 and P22 attachment sites. Primary structure and interaction with Escherichia coli integration host factor.
Although the lambdoid bacteriophage phi 80 and P22 possess site-specific recombination systems analogous to bacteriophage lambda, they have different attachment (att) site specificities. We have identified and determined the nucleotide sequences of the att sites of phi 80 and P22 and have examined the interaction of these sites with purified Escherichia coli integration host factor (IHF). The sizes of the homologous core regions of the att sites vary greatly: P22 has a 46-base pair core, while phi 80 and lambda have 17- and 15-base pair cores, respectively. The core sequences of the three phage show no significant homology, although dispersed regions of homology in arm sequences indicate that the three phage att sites are related. All three att sites have a high A + T composition, and restriction fragments carrying these sites migrate anomalously upon polyacrylamide gel electrophoresis. IHF binds to a site to the left of the common core in the phi 80 and P22 phage att sites (attP) and to a site to the right of the core in P22 attP and attB (the bacterial att site). In the lambda system, IHF interacts with three regions on attP (designated H1, H2, and H') and none on attB (Craig N., and Nash, H.A. (1984) Cell 39, 707-716). Alignment of the IHF sites of all three phage results in a consensus sequence for IHF binding, Pyr-AANNNNTTGATAT. Among the three phage, the number of IHF sites differs; however, the location and orientation of the binding sites in relation to the respective core regions are well conserved. An IHF site analogous to lambda H2 is present in both phi 80 and P22 attP, while a site analogous to lambda H' is present in P22 attP. This conservation suggests that IHF plays a very similar role in the site-specific recombination pathways of all three phage, and that the flanking arm sequences are necessary for phi 80 and P22 attP function, as is the case for lambda attP function. These structural similarities presumably reflect a conservation of the mechanism of site-specific recombination for the three phage.
Bacteriophage lambda↗
Generation of single base-pair deletions, insertions, and substitutions by a site-specific recombination system.
The sequence analysis of both products of individual phi 80 site-specific recombination events in vivo shows that recombination with a secondary attachment (att) site generates several different novel joints at the mismatched position: one recombination event resulted in a single base-pair deletion and two other recombination events resulted in two different single base-pair substitutions. The characterized products of recombination can be straightforwardly interpreted as the outcome of strand exchange involving staggered nicks bracketing the heterology within an overlap region of five to nine base pairs. In comparison, more complex segregation patterns have been observed in previous studies of lambda recombination between nonidentical att sites; the nature of the overlap region heterology may have a significant effect on the segregation patterns. To recover both products of a single recombination event, we used a plasmid that carries the phi 80 int and xis genes and both att sites. Because the two att sites are situated in opposite orientation, intramolecular recombination between them inverts rather than deletes the intervening segment of DNA. Although subsequent reinversion restores the original gross genetic arrangement, single base-pair insertions, deletions, and substitutions are introduced at the sites of recombination. One of the mutations improves the recombination efficiency of the secondary att site and thereby converts a formerly "stable" sequence to an efficient target for rearrangement, and other mutations are predicted to alter the specificity of recombination. These pathways may also provide useful models for the efficient generation of localized sequence diversity on a development (as well as evolutionary) time scale.
Bacteriophage lambda↗
Interaction of the lambda site-specific recombination protein Xis with attachment site DNA.
Nuclease protection experiments show that Xis protein of bacteriophage lambda specifically binds attachment (att) site DNA. The region of Xis binding, present in both the phage att site and the right prophage att site, extends from position -102 to position -62 in the P arm. The sequence of this region, the positions of purines protected by Xis against methylation, and the binding of Xis to a resected att site indicate the presence of two binding sites. The postulated recognition elements, contained in 13-base-pair direct repeats separated by 7 base pairs, are situated on the same face of the DNA helix. Protection experiments performed with DNase I suggest that the DNA wraps around (or along the surface of) the bound Xis protein. The Xis binding data presented here establishes that Xis, like the other two proteins involved in lambda site-specific recombination, interacts specifically with att DNA. This rules out that class of models in which the profound effects of Xis on the directionality of site-specific recombination are mediated solely through protein-protein interactions or modification of another protein. In addition, nuclease protection experiments with pairwise combinations of the proteins show that Xis and integration host factor (IHF), or Xis and Int, can bind simultaneously to either the phage or right prophage att sites, and the DNA sequences protected are the sum of those protected with each protein alone. It is therefore unlikely that the effect of Xis on the direction of recombination is exerted by directly blocking the binding of Int or IHF to one or more of their respective binding sites.
Attachment Sites, Microbiological↗
Determinants of directionality in lambda site-specific recombination.
The DNA structural features governing directionality in lambda site-specific recombination are shown to reside in regions of the phage attachment site more than 70 bp to the left and more than 40 bp to the right of the cross-over region. Disposition of these sequences on the same attachment site in integration, and on different attachment sites in excision, determines the opposite effects of Xis protein upon the two reactions (stimulation of excision and inhibition of integration). The binding of Xis to two adjacent directly repeated sequences in the left phage arm is shown to occur in a highly cooperative manner, to alter the conformation of the DNA, and to produce a 32-fold stimulation of Int binding to an adjacent locus.
Bacteriophage lambda↗
Site-specific recombination systems of phages phi 80 and P22: binding sites of integration host factor and recombination-induced mutations.
Explore the source record for details and available documents.
Base Sequence↗
Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda.
Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.
Attachment Sites, Microbiological↗