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A Landy

Publications and source records attributed to A Landy.

At least 37 records · Page 2Linked to original sources
PubMed1991

A switch in the formation of alternative DNA loops modulates lambda site-specific recombination.

The virally encoded Xis protein is one of the components in the site-specific recombination reactions of bacteriophage lambda. It is required for excisive recombination and inhibits integrative recombination. The mechanism of Xis inhibition of the integration reaction was investigated by methylation protection assays (footprinting analyses) in conjunction with recombination assays. Xis is shown to mediate the formation of a specific attP looped structure involving cooperative and competitive long-range interactions among integrase, integration host factor, and Xis proteins. This higher-order structure precludes supercoiled attP from engaging in the productive partner interactions that lead to execution of the first strand exchange in integrative recombination. In addition to its previously characterized role in excision, Xis-induced DNA bending is postulated to act as a regulatory switch (in an alternative loop mechanism) that converts the attP intasome from an integrative-competent complex to a nonreactive one.

Bacterial Proteins↗
PubMed1990

Mapping of a higher order protein-DNA complex: two kinds of long-range interactions in lambda attL.

To map the protein-protein and protein-DNA interactions involved in lambda site-specific recombination, Int cleavage assays with suicide substrates, nuclease protection patterns, gel retardation experiments, and quantitative Western blotting were applied to wild-type attL and attL mutants. The results lead to a model in which one IHF molecule bends the attL DNA and forms a higher order complex with the three bivalent Int molecules required for excisive recombination. It is proposed that each of the Int molecules binds in a unique manner: one bridges two DNA binding sites in cis, one is held via its high affinity amino-terminal DNA binding domain, and the third depends upon protein-protein interactions in addition to its low affinity carboxy-terminal DNA binding domain. This protein-DNA complex contains two unsatisfied DNA binding domains, each with a different sequence specificity, and is well suited to specific interactions with an appropriate recombination partner.

Bacteriophage lambda↗
PubMed1990

Interactions between lambda Int molecules bound to sites in the region of strand exchange are required for efficient Holliday junction resolution.

lambda Site-specific recombination proceeds via two sequential single-strand exchanges that first generate and then resolve a Holliday recombination intermediate. The resolution of artificial Holliday junctions (chi-forms) is well suited to studying the mechanisms involved in reciprocal strand exchange because the linear products of this reaction are stable and easily quantitated. To study the interactions between Int molecules bound at the sites of strand exchange, artificial Holliday junctions containing only the seven base-pair overlap region and the four core-type Int binding sites were used as a model system. In vitro resolution of these structures yields products of both top- and bottom-strand exchange. An abortive product resulting from simultaneous cleavage of the top and bottom strands also occurs at low frequency. Inactivation of one of the four Int binding sites by multiple base substitutions does not significantly affect the efficiency of resolution but has a dramatic effect on the directionality, i.e. the choice of top- or bottom-strand exchange. When any two of the four core-type sites are similarly inactivated, strand exchange is very inefficient and the amount of aberrant cleavage is somewhat greater than for the Holliday junction with four intact Int binding sites. Analysis of the resolution products of Holliday junctions with various combinations of defective Int binding sites leads to the following conclusions: (1) three functional core-type Int binding sites are necessary and sufficient for a strand exchange; (2) the Int molecules that are partners in a strand exchange interact with Int bound to a "cross-core" site that is not directly involved in carrying out the reaction; (3) Int molecules bound to the core-type sites interact in a way that reduces the occurrence of abortive double-strand cleavage events.

Bacteriophage lambda↗
PubMed1989

Half-att site substrates reveal the homology independence and minimal protein requirements for productive synapsis in lambda excisive recombination.

The early events in site-specific excisive recombination were studied with phage lambda half-att sites that have no DNA to one side of the strand exchange region; they carry a single core-type integrase binding site and either P or P' arm flanking DNA. These half-attR and half-attL sites exhibit normal properties for the initial (covalent) top-strand transfer and form stable intermediates independent of later steps in the reaction. With these novel substrates we show that Xis specifically promotes the first strand exchange and that attL enhances Int cleavage at the top-strand site of attR. It is also shown that synapsis and initial strand transfers do not require DNA-DNA pairing but are mediated by protein-protein and protein-DNA interactions. These involve the two top-strand Int binding sites (required for the first strand exchange) and, in addition, one of the two bottom-strand sites (C') responsible for the second strand exchange.

Attachment Sites, Microbiological↗
PubMed1989

Phasing of protein-induced DNA bends in a recombination complex.

Many of the structures responsible for replication, transcription initiation and recombination arise from complex sets of protein-protein interactions and the folding of DNA in three dimensions, with protein-induced bending of DNA often playing an integral role. The magnitude and orientation of DNA bending induced by various single proteins has been estimated by gel mobility shift methods and by modelling of crystallographic data. The site-specific recombination by which bacteriophage lambda (phage lambda) integrates into the chromosome of its host Escherichia coli requires a host protein, 'integration host factor' (IHF), which is known to be able to bend the DNA to which it binds. To determine the three-dimensional path of DNA within the higher order structure responsible for phage lambda site-specific recombination, we have determined the relative direction of IHF-induced bending at each of the three binding sites within the complex. IHF, which appears to bend DNA by more than 140 degrees, is a major determinant of the DNA path in the recombination complex and is also involved in a wide range of other cellular events.

Bacterial Proteins↗
PubMed1989

DNA looping generated by DNA bending protein IHF and the two domains of lambda integrase.

The multiprotein-DNA complexes that participate in bacteriophage lambda site-specific recombination were used to study the combined effect of protein-induced bending and protein-mediated looping of DNA. The protein integrase (Int) is a monomer with two autonomous DNA binding domains of different sequence specificity. Stimulation of Int binding and cleavage at the low affinity core-type DNA sites required interactions with the high affinity arm-type sites and depended on simultaneous binding of the sequence-specific DNA bending protein IHF (integration host factor). The bivalent DNA binding protein is positioned at high affinity sites and directed, by a DNA bending protein, to interactions with distant lower affinity sites. Assembly of this complex is independent of protein-protein interactions.

Bacterial Proteins↗
PubMed1989

Rho-dependent transcription termination in the tyrT operon of Escherichia coli.

A 178-bp repeat sequence comprises the distal end of the tyrT operon and also encodes the signal for Rho-dependent transcription termination. It is shown here that Rho-dependent transcription termination is highly efficient for each of the individually cloned first and second repeats. The presence of either repeat results in a termination efficiency of 88% (12% readthrough). When an individual DNA repeat is shortened, starting from the promoter-proximal end, so that only 56 bp remain upstream from the termination site, Rho-dependent termination activity is reduced approximately three-fold (to give 38% readthrough) and with further shortening, so that only 15 bp remain upstream, Rho-dependent termination is reduced an additional 1.8-fold (to give 59% readthrough). Finally, it is shown that the presence or absence of the tyrT terminator upstream from the lambda tR1 terminator does not affect the efficiency of transcription at termination lambda tr1.

Base Sequence↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1988

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1987

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↗
PubMed1986

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↗
PubMed1986

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↗
PubMed1986

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↗