On the role of the bacteriophage lambda int gene product in site specific recombination

Integrative recombination of phage λ DNA occurs in extracts made from cells synthesizing int protein. In this paper we show that extracts of cells containing temperature-sensitive int protein are inactivated more rapidly by incubation at 38 °C than are wild-type extracts. This indicates that the int protein is directly involved in the recombination reaction.     

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.     

The form of the DNA substrate required for excisive recombination of bacteriophage lambda.

We have studied the excision reaction of bacteriophage lambda, both in vivo and in vitro, using as a substrate a λatt²(L × R) phage carrying both the right and left-hand prophage attachment sites. Int and Xis are provided by induction of the heat-inducible defective prophage, λc1857 ΔH1. After a brief induction (5 min) of these cells, excisive recombination is blocked in the presence of the DNA gyrase inhibitor, coumermycin. However, after a longer induction (greater than 30 min) excisive recombination occurs efficiently under conditions where λ integrative recombination is inhibited by coumermycin. In such extensively induced coumermycin-treated cells, infecting λatt²(L × R) DNA is not supercoiled, and recombinants are found among the relaxed covalently closed circular DNA.In vitro, starting with a hydrogen-bonded λatt² DNA substrate, excision is insensitive to high concentrations of coumermycin and novobiocin. To study the DNA substrate requirements for excisive recombination in more detail, we have developed a restriction fragment assay for excisive recombination. With this assay, we demonstrate that supercoiled, hydrogen-bonded, and linear λatt² DNA molecules are all efficient substrates in the in vitro excision reaction. Spermidine is required but ATP and Mg²⁺ are not. We conclude that supercoiling is not an absolute requirement for site-specific recombination of λ.     

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.     

Interaction of int protein with specific sites on lambda att DNA.

We have studied the interaction of highly purified Int protein with DNA restriction fragments from the lambda phage attachment site (attP) region. Two different DNA sequences are protected by bound Int protein against partial digestion by either pancreatic DNAase or neocarzinostatin. One Int binding site includes the 15 bp common core sequence (the crossover region for site-specific recombination) plus several bases of sequence adjoining the core in both the P and P' arms. The second Int-protected site occurs 70 bp to the right of the common core in the P' arm, just at the distal end of the sequence encoding Int protein. The two Int binding sites are of comparable size, 30-35 bp, but do not share any extensive sequence homology. The interaction of Int with the two sites is distinctly different, as defined by the observation that only the site in the P' arm and not the site at the common core region is protected by Int in the face of challenge by the polyanion heparin. Restriction fragments containing DNA from the bacterial attachment site (attB) region exhibit a different pattern of interaction with Int. In the absence of heparin, a smaller (15 bp) sequence, which includes the left half of the common core region and the common core-B arm juncture, is protected against nuclease digestion by Int protein. No sequences from this region are protected by Int in the presence of heparin.     

The Nul subunit of bacteriophage lambda terminase binds to specific sites in cos DNA.

The maturation and packaging of bacteriophage lambda DNA are under the control of the multifunctional viral terminase enzyme, which is composed of the protein products of Nu1 and A, the two most leftward genes of the phage chromosome. Terminase binds selectively to the cohesive end site (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12-base single-stranded cohesive ends of the mature phage genome. The purified gpNu1 subunit of terminase forms specific complexes with cos lambda DNA. DNase I footprinting experiments showed that gpNu1 bound to three distinct regions near the extreme left end of the lambda chromosome. These regions coincided with two 16-base-pair sequences (CTGTCGTTTCCTTTCT) that were in inverted orientation, as well as a truncated version of this sequence. Bear et al. (J. Virol. 52:966-972,1984) isolated a mutant phage which contained a CG to TA transition at the 10th position of the rightmost 16-base-pair sequence, and this phage (termed lambda cos 154) exhibits a defect in DNA maturation when it replicates in Escherichia coli which is deficient in integration host factor. Footprinting experiments with cos 154 DNA showed that gpNu1 could not bind to the site which contained the mutation but could protect the other two sites. Since the DNA-packaging specificity of terminase resides in the gpNu1 subunit, these studies suggest that terminase uses these three sites as recognition sequences for specific binding to cos lambda.     

The EcoRI restriction endonuclease with bacteriophage lambda DNA. Equilibrium binding studies.

The EcoRI restriction endonuclease was found by the filter binding technique to form stable complexes, in the absence of Mg2+, with the DNA from derivatives of bacteriophage lambda that either contain or lack EcoRI recognition sites. The amount of complex formed at different enzyme concentrations followed a hyperbolic equilibrium-binding curve with DNA molecules containing EcoRI recognition sites, but a sigmoidal equilibrium-binding curve was obtained with a DNA molecule lacking EcoRI recognition sites. The EcoRI enzyme displayed the same affinity for individual recognition sites on lambda DNA, even under conditions where it cleaves these sites at different rates. The binding of the enzyme to a DNA molecule lacking EcoRI sites was decreased by Mg2+. These observations indicate that (a) the EcoRI restriction enzyme binds preferentially to its recognition site on DNA, and that different reaction rates at different recognition sites are due to the rate of breakdown of this complex; (b) the enzyme also binds to other DNA sequences, but that two molecules of enzyme, in a different protein conformation, are involved in the formation of the complex at non-specific consequences; (c) the different affinities of the enzyme for the recognition site and for other sequences on DNA, coupled with the different protein conformations, account for the specificity of this enzyme for the cleavage of DNA at this recognition site; (d) the decrease in the affinity of the enzyme for DNA, caused by Mg2+, liberates binding energy from the DNA-protein complex that can be used in the catalytic reaction.     

The Fi-gene product of bacteriophage lambda. Purification and properties

The FI gene product of bacteriophage lambda has been purified extensively using a biochemical assay that measures assembly of lambda phage particles in vitro. The molecular weight of the native protein was estimated to be 21700 with an S20, w of 2.1 S and a Stokes radius of 2.5 nm. The molecular weight in dodecylsulfate was estimated to be 19000. The protein is highly acidic with an isoelectric point less than 4.1.     

The R gene product of bacteriophage lambda is the murein transglycosylase

Summary The radioactively labeled proteins synthesised in Escherichia coli minicells infected by bacteriophage R and R ⁺ were compared by polyacrylamide gel electrophoresis. R mutants, which have lost the ability to lyse host cells, lack a polypeptide of molecular weight 17.5 kD corresponding to the molecular weight of murein transglycosylase — a bacteriolytic enzyme from lysates which we have described previously. It has been shown by direct comparison using radio-labeled enzyme that transglycosylase comigrates with the R gene product. The enzyme was endetectable in induced cultures of E. coli W3350 su ^o (cI857 Ram5) and C600 (cI857 acR301), while it was present in a R _– ⁺ mutant lysate. We conclude that the transglycosylase is the R gene product.Abbreviations Muropeptide CA GlcNac-1-4-1,6-anhydro-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide CB GlcNac-MurNac-GlcNac-1,6-anhydro-MurNac in which the carboxyl groups of MurNac and 1,6-anhydro-MurNac are substituted by the tetrapeptide L-Ala-D-Glu-msA₂pm-D-Ala - muropeptide C3 dimer of the two units GlcNac-MurNac-L-Ala-D-Glu-msA₂pm-D-Ala which are connected by D-D peptide bond between D-Ala and msA₂pm - GlcNac N-acetyl-D-glucosamine - MurNac N-acetylmuramic acid - msA₂pm meso-diaminopimelic acid - rivanol 6,9-diamino-2-ethoxyacridine lactate - SDS sodium dodecyl sulfate     

Partially deficient methylation of cytosine in DNA at CCATGG sites stimulates genetic recombination of bacteriophage lambda

Lambda bacteriophages grown on arl mutants of Escherichia coli ("Arl-" phages) display intermediate levels of cytosine methylation: less 5-methylcytosine (m5C) than phages grown on wild-type bacteria ("Arl+" phages) but more than phages grown on dcm mutants, and thus lacking the methylated sequences (Cm5CATGG) characteristic of E. coli K-12 bacteria ("Dcm-" phages). "Arl-" phages are one twelfth as resistant to Eco RII restriction (recognition site CCATGG) as "Arl+" phages, but 40-fold more resistant than "Dcm-" phages. Chromatographic analyses show the 5-methylcytosine content of "Arl-" DNA to be one third that of "Arl+" DNA. Altered cytosine methylation frequency correlates with two previously described properties of "Arl-" phages, increased genetic recombination and unusual sensitivity of phage DNA to endonuclease S1, which are absent in phages grown on dcm or dcm arl bacteria. Methylated/unmethylated heteroduplex DNA prepared in vitro (one strand from Eco RII-modified phages/one from "Dcm-" phages) is highly recombinogenic but not S1-sensitive. We hypothesize that hemimethylated CCATGG sites in "Arl-" DNA are necessary and sufficient for enhanced recombination, and necessary but not sufficient for S1 sensitivity.     

DNA without supertwists can be an in vitro substrate for site-specific recombination of bacteriophage lambda.

A negatively supertwisted substrate is required for site-specific recombination of baeteriophage λ in vitro under conditions of high ionic strength. This requirement is eliminated under conditions of low ionic strength. Both integrative (attB × attP) and excisive (attR × attL) recombinations display this property. Either nicked or hydrogen-bonded circular forms of DNA recombine in low ionic strength environments in the absence of DNA gyrase activity.     

Exclusion of bacteriophage T1 by bacteriophage lambda. I. Early exclusion requires lambda N gene product and host factors involved in N gene expression.

Two modes of exclusion of T1 by lambda are distinguished. "Early" exclusion depends on gene N, but not on gene Q. It is at least partially ineffective against T1am23. "Late" exclusion depends on gene Q and effects T1am23 as well as T1+. Early exclusion is a direct effect of N gene product, rather than N gene being required for the expression of some other lambda gene. Three host mutations, groN, nusA, and nusB, known to interfere with lambda replication by affecting N gene expression, also interfere with the ability of lambda to exclude T1.     

Joint molecules of lambda DNA as an intermediate of genetic recombination.

Summary Joint molecules of λ DNA formed in the absence of DNA replication, which may be involved in the process of genetic recombination can be observed as branched DNA derived from different phage particles. These molecules are associated through base-pair hydrogen bonding in synaptic regions, usually with short single-stranded gaps. Furthermore, joint molecules could be accumulated up to ten fold when λ was irradiated with ultraviolet light before infection ofpolI mutant ofE. coli. Infection at low multiplicity did not give rise to joint molecules. These results suggest that single-strand breaks and gaps introduced in duplex λ DNA facilitate the formation of joint molecules.