The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.     

Lysis protein S of phage lambda functions in Saccharomyces cerevisiae.

The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.     

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.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

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.     

Ribosomal protein S18 identified as a cofilin-binding protein by using phage display library

We previously reported that an actin-binding protein, cofilin, is involved in superoxide production, phagocytosis, and chemotaxis in activated phagocytes through cytoskeletal reorganization. To elucidate the functions of cofilin in greater detail we tried to identify cofilin-binding proteins by using a phage-displayed cDNA library constructed from human brain mRNAs. Several phage clones capable of binding to cofilin were obtained, and the phage with the strongest binding affinity contained the C-terminal half of ribosomal protein S18. To confirm the interaction between the S18 protein and cofilin, we investigated whether cofilin would bind to His-tagged S18 protein immobilized in Ni-NTA-agarose gel. Cofilin and the S18 protein co-eluted with a low pH (4.5) buffer, suggesting that the proteins interact with each other. Preincubation of cofilin with actin abrogated the binding to protein S18, indicating that cofilin interacts with S18 protein at the actin-binding site, and cofilin co-immunoprecipitated with FLAG-tagged S18 protein expressed in COS-7 cells. These results suggest that some cofilin molecules bind the ribosomal S18 protein under physiological conditions.     

Ribosomal components required for binding protein S1 to the 30 S subunit of Escherichia coli.

30 S subunits of Escherichia coli ribosomes washed with 3 m-NH₄C1 lose proteins S2, S3, S9, S10, S14, S20 and S21, as well as their ability to bind S1 with high affinity (Laughrea and Moore, 1978). Binding activity is restored when the split proteins are added back to the protein-deficient cores. Here we show that, among the split proteins, S9 is by far the most effective in restoring S1 binding capability to 3 m-NH₄Cl cores.     

IHF stimulation of lambda cII gene expression is inhibited by the E. coli NusA protein

The effects of E. coli proteins Integrative Host Factor (IHF) and NusA on the regulation of lambda cII gene expression are presented. As reported previously (Peacock et al. [1984] Proc. Natl. Acad. Sci. USA 81, 6009-6013), IHF stimulates the DNA-directed in vitro synthesis of cII protein or its first dipeptide, fMet-Val. Whereas NusA, by itself, has no effect on cII expression, the presence of NusA inhibits the IHF-mediated stimulation of cII synthesis.     

Domain structure of gpNu1, a phage lambda DNA packaging protein

The terminase enzyme from bacteriophage lambda is responsible for the insertion of a dsDNA genome into the confines of the viral capsid. The holoenzyme is composed of gpA and gpNu1 subunits in a gpA(1) x gpNu1(2) stoichiometry. While genetic studies have described regions within the two proteins responsible for DNA binding, capsid binding, and subunit interactions in the holoenzyme complex, biochemical characterization of these domains is limited. We have previously described the cloning, expression, and biochemical characterization of a soluble DNA binding domain of the terminase gpNu1 subunit (Met1 to Lys100) and suggested that the hydrophobic region spanning Lys100 to Pro141 defines a domain responsible for self-association interactions, and that is important for cooperative DNA binding [Yang et al. (1999) Biochemistry 38, 465-477]. We further suggested that the genetically defined gpA-interactive domain in the C-terminal half of the protein is limited to the C-terminal approximately 40 amino acids of gpNu1. Here we describe the cloning, expression, and biochemical characterization of gpNu1DeltaP141, a deletion mutant of gpNu1 that comprises the DNA binding domain and the putative hydrophobic self-assembly domain of the full-length protein. Purified gpNu1DeltaP141 shows a strong tendency to aggregate in solution; However, the protein remains soluble in 0.4 M guanidine hydrochloride, and circular dichroism (CD) and fluorescence spectroscopic studies demonstrate that the protein is folded under these conditions. Moreover, CD spectroscopy and thermally induced unfolding studies suggest that the DNA binding domain and the self-association domain represent independent folding domains of gpNu1DeltaP141. The mutant protein interacts weakly with the gpA subunit, but does not form a catalytically competent holoenzyme complex, suggesting that the C-terminal 40 residues are important for appropriate subunit interactions. Importantly, gpNu1DeltaP141 binds DNA tightly, but with less specificity than does full-length protein, and the data suggest that the C-terminal residues are further required for specific DNA binding activity. The implications of these results in the assembly of a functional holoenzyme complex are discussed.     

Reversion of Q beta RNA phage mutants by homologous RNA recombination.

Q beta phage RNAs with inactivating insertion (8-base) or deletion (17-base) mutations within their replicase genes were prepared from modified Q beta cDNAs and transfected into Escherichia coli spheroplasts containing Q beta replicase provided in trans by a resident plasmid. Replicase-defective (Rep-) Q beta phage produced by these spheroplasts were detected as normal-sized plaques on lawns of cells containing plasmid-derived Q beta replicase, but were unable to form plaques on cells lacking this plasmid. When individual Rep- phage were isolated and grown to high titer in cells containing plasmid-derived Q beta replicase, revertant (Rep+) Q beta phage were obtained at a frequency of ca. 10(-8). To investigate the mechanism of this reversion, a point mutation was placed into the plasmid-derived Q beta replicase gene by site-directed mutagenesis. Q beta mutants amplified on cells containing the resultant plasmid also yielded Rep+ revertants. Genomic RNA was isolated from several of the latter phage revertants and sequenced. Results showed that the original mutation (insertion or deletion) was no longer present in the phage revertants but that the marker mutation placed into the plasmid was now present in the genomic RNAs, indicating that recombination was one mechanism involved in the reversion of the Q beta mutants. Further experiments demonstrated that the 3' noncoding region of the plasmid-derived replicase gene was necessary for the reversion-recombination of the deletion mutant, whereas this region was not required for reversion or recombination of the insertion mutant. Results are discussed in terms of a template-switching model of RNA recombination involving Q beta replicase, the mutant phage genome, and plasmid-derived replicase mRNA.     

Biochemical analysis of att-defective mutants of the phage lambda site-specific recombination system

Three mutations previously mapped to the common core region of the bacteriophage lambda att site have been sequenced. All were found to be due to the deletion of a T residue from a string of six T residues within the 15 base-pair core, the region of homology between the recombining sites. As judged by DNAase I protection experiments, binding of the Int protein is the same in the mutant and wild-type core sites. However, a difference in the Int binding to mutant cores is observed when the small neocarzinostatin molecule is used as a nuclease probe. The differences between mutant and wild type lead to the suggestion that Int is interacting with sequences at the core-arm junctions. Accordingly, the mutants are proposed to be defective in the spacing of Int monomers bound at two recognition sequences spanning the core-arm junctions. The anomalous electrophoretic mobility of wild-type att fragments and, more specifically, the effect of the single base core deletion on electrophoretic mobility are discussed in the text and in the Appendix. The mutant att²501, defective in both att and int functions, was sequenced and found to be a 335 base-pair deletion removing the coding region for 25 amino acids from the carboxy-terminal end of Int, as well as the entire att site. The postulated origin of the 501 mutation is also consistent with the model of two juxtaposed Int recognition sites.