Elements involved in site-specific recombination in bacteriophage lambda

doi:10.1016/0022-2836(75)90275-2

Journal of Molecular Biology

Volume 91, Issue 4, 5 February 1975, Pages 489-499

https://doi.org/10.1016/0022-2836(75)90275-2 Get rights and content

Abstract

A sensitive assay for bacteriophage lambda site-specific recombination has been developed using the λatt² phage described by Shulman & Gottesman (1971). This large EDTA-sensitive transducing phage, which carries both the left and right prophage attachment sites, undergoes an Int and Xis-dependent excision reaction to yield a smaller EDTA-resistant non-transducing phage bearing a phage attachment site.

We have used this system to study the characteristics of site-specific excision in vivo. We find that: (1) the kinetics of excision are rapid; (2) inhibition of macromolecular processes such as transcription, translation, and DNA replication does not affect the extent of the excisional recombination.

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News from the protein mutability landscape
2013, Journal of Molecular Biology
Citation Excerpt :
Comprehensive experimental mutagenesis studies confirmed that the effect of point mutations (SAAS) upon function depends crucially on their positions in the protein sequence [59–61]. Even within a unit as familiar as the DNA binding domain of the Escherichia coli LacI [62,63] repressor, almost any variant can be tolerated at some positions while, at others, all variants affect function (Fig. 2a). Simple structural constraints might suffice to explain this variability: to accommodate the negatively charged DNA, binding regions of the repressor contain positively charged residues.
Some mutations of protein residues matter more than others, and these are often conserved evolutionarily. The explosion of deep sequencing and genotyping increasingly requires the distinction between effect and neutral variants. The simplest approach predicts all mutations of conserved residues to have an effect; however, this works poorly, at best. Many computational tools that are optimized to predict the impact of point mutations provide more detail. Here, we expand the perspective from the view of single variants to the level of sketching the entire mutability landscape. This landscape is defined by the impact of substituting every residue at each position in a protein by each of the 19 non-native amino acids. We review some of the powerful conclusions about protein function, stability and their robustness to mutation that can be drawn from such an analysis. Large-scale experimental and computational mutagenesis experiments are increasingly furthering our understanding of protein function and of the genotype–phenotype associations. We also discuss how these can be used to improve predictions of protein function and pathogenicity of missense variants.
Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage λ
1983, Journal of Molecular Biology
Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage λ attachment site by inserting the phage chromosome into and then excisiing 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 λ 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.
Site-specific recombination. Xis-independent excisive recombination of bacteriophage lambda
1981, Journal of Molecular Biology
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.
The role of himA and xis in lambda site-specific recombination
1980, Journal of Molecular Biology
We have examined the requirements for integrative and excisive recombination of bacteriophage γ, by providing large quantities of Int and varying amounts of Xis in himA⁺ and himA mutant cells. The wild type himA gene product is not essential for excisive recombination in the presence of large amounts of Xis. When Xis is limiting or missing, however, the himA gene product plays a major role in excision. Similarly, integrative recombination is normally quite dependent on himA product. Limited integrative recombination can occur in a himA mutant, but only in the presence of large quantities of Xis. We propose that alternative pathways exist for these two reactions; one pathway (int, Him A) is favored for integration but can be used for excision, while the other (Int, Xis) is highly efficient for excisive recombination but can also be used for integrative recombination.
The form of the DNA substrate required for excisive recombination of bacteriophage λ
1979, Journal of Molecular Biology
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.
Structural features of λ site-specific recombination at a secondary att site in galT
1979, Cell
Integration of bacteriophage λ DNA into the chromosome of its E. coli host proceeds via a site-specific recombination between specific loci (att sites) on the phage and bacterial chromosomes. Infection of an E. coli host deleted for the primary bacterial att site results in λ integration with reduced efficiency at a number of different “secondary att sites” scattered around the E. coli chromosome. The first DNA sequence analysis of such a secondary att site, that occurring in the galT gene, is reported here, and several features pertinent to the mechanism of int-dependent site-specific recombination are discussed.
Previous studies have shown that the crossover in int-dependent recombination must be somewhere within a 15 bp sequence (core region) common to the phage and primary bacterial att sites, as well as to the left and right prophage att sites which are at the junctures between prophage and host DNA. Comparison of the galT secondary prophage att sites with the primary prophage att sites allows determination of the analogous “core” region in the galT secondary att site. The 15 bp sequence thus identified shows an interrupted homology (8 out of 15) with the wild-type core. The extent and arrangement of nonhomologous bases allow precise placement of the crossover point for this recombination to the +4–+5 internucleotide bond of the core region.
Sequences flanking the core region show no obvious homology with analogous sequences of the phage or primary bacterial att sites. Comparison of the galT left prophage att site with the analogous wild-type site is of particular interest and is discussed in relation to binding studies with purified int protein.

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^☆: One of us (S. G.) was a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research while this work was done.

^†: Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139, U.S.A.

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Journal of Molecular Biology

Elements involved in site-specific recombination in bacteriophage lambda☆

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