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.     

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.     

Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

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 recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Transfer of a bacterial gene using phage lambda transfecting DNA]

A new amber mutation of phage with the gene coding synthesis of beta-galactosidase was received by recombination. With the help of transfection DNA isolated from this phage the transfer of the gene coding the beta-galactosidase synthesis to the recipient phage-resistant E. coli cell was realized. The suggested model can be used for the gene transfer to the recipient phage-resistant cells or other species of bacteria with transfection DNA.     

Repair mechanisms involved in prophage reactivation and UV reactivation of UV-irradiated phage lambda

Survival of UV-irradiated phage λ is increased when the host is lysogenic for a homologous heteroimmune prophage such as λimm434 (prophage reactivation). Survival can also be increased by UV-irradiating slightly the non-lysogenic host (UV reactivation).Experiments on prophage reactivation were aimed at evaluating, in this recombination process, the respective roles of phage and bacterial genes as well as that of the extent of homology between phage and prophage.To test whether UV reactivation was dependent upon recombination between the UV-damaged phage and cellular DNAs, lysogenic host cells were employed. Such hosts had thus as much DNA homologous to the infecting phage as can be attained. Therefore, if recombination between phage and host DNAs was involved in this repair process, it could clearly be evidenced.By using unexposed or UV-exposed host cells of the same type, prophage reactivation and UV reactivation could be compared in the same genetic background.The following results were obtained: (1) Prophage reactivation is strongly decreased in a host carrying recA mutations but quite unaffected by mutation lex-I known to prevent UV reactivation; (2) In the absence of the recA⁺ function, the red⁺ but not the int⁺ function can substitute for recA⁺ to produce prophage reactivation, although less efficiently; (3) Prophage reactivation is dependent upon the number of prophages in the cell and upon their degree of homology to the infecting phage. The presence in a recA host of two prophages either in cis (on the chromosome) or in trans (on the chromosome and on an episome) increases the efficiency of prophage reactivation; (4) Upon prophage reactivation there is a high rate of recombination between phage and prophage but no phage mutagenesis; (5) The rate of recombination between phage and prophage decreases if the host has been UV-irradiated whereas the overall efficiency of repair is increased. Under these conditions UV reactivation of the phage occurs as in a non-lysogen, as attested by the high rate of mutagenesis of the restored phage.These results demonstrate that UV reactivation is certainty not dependent upon recombination between two pre-existing DNA duplexes. The hypothesis is offered that UV reactivation involves a repair mechanism different from excision and recombination repair processes.     

Role of genes O and P in the replication of bacteriophage lambda DNA.

DNA replication in coliphage λ occurs in two stages. The first round of replication generates mainly circular progeny DNA by a double-branched θ-type replicative form (Ogawa et al., 1968; Schnös &; Inman, 1970). In the late stage of λ DNA replication, however, σ-type rolling-circle replicative form DNA molecules, which produce multigenomic linear concatemers, are primarily found (Takahashi, 1974).At both early and later times, a temperature shift of λ Ots or Pts infected cells from 32 °C (permissive) to 43 °C (non-permissive temperature) caused a rapid reduction of the rate of radioactive precursor incorporation into λ DNA, showing that the gene O and P products are essential for the continuation of λ DNA synthesis. Observations on the molecular fine structure of the replicating fork after a temperature shift revealed characteristic long “single-strand connections” and single-strand “whiskers” at the branch point. These observations suggest that λ gene O and P products are directly involved in the propagation of daughter strands.     

Filter-supported preparation of lambda phage DNA

A rapid and simple method is described for the isolation of DNA from phage lambda which requires neither special equipment nor expensive material such as cesium chloride for ultracentrifugation nor extractions with organic solvents or ethanol precipitation. Microgram quantities of lambda DNA are obtained in less than 2 h from 90-mm plate lysates or 5-ml liquid cultures. The method allows the simultaneous isolation of large numbers of probes, e.g., clones from phage libraries. Lambda phages are precipitated by polyethylene glycol/sodium chloride and recovered by low speed centrifugation onto glass fiber filters positioned in disposable syringes. The DNA of phages is released by a 50% formamide/4 M sodium perchlorate solution, washed in filter-bound form, eluted with a small volume of low-salt buffer or water, and finally recovered by centrifugation. Comparison of the DNA isolated by this method with that obtained by two conventional procedures reveals both a similar recovery and a similar suitability for restriction enzyme digestion and subcloning.     

Rapid isolation of phage lambda DNA

A modified procedure in two versions (micro, for 10 ml of phage lysate, and macro, for 200-500 ml) is described for preparing lambda phage DNA. The main advantage of the modified method is that it gives a possibility to isolate high-quality DNA from lambda phage lysates in 2-3 hrs. Only standard solutions (TE, NaCl, SDS, MgCl2, EDTA, RNAse A) were used throughout the whole protocol. Incubation with DNAse I and proteinase K was omitted and in microvariant concentration of the phage by PEG 6000 was excluded. Digestion by RNAse A was performed in solution with EDTA and SDS and leads to RNA degradation. The yields of DNA (0.5-2 micrograms per ml of L-broth) are similar to those obtained by other methods. DNA quality is better than in the samples of DNA prepared by other express-methods and practically the same as after CsCl centrifugation. DNA can be used for splitting by restriction enzymes, cloning and gene library construction.     

Isolation and genetic analysis of bacterial mutations gpr blocking the replication of various lambda phages

Bacterial mutations affecting phi 80 DNA replication have been isolated and designated gpr2, 27. The main difference between gpr2, 27 and groP, grp mutations described earlier is that mutations gpr2, 27 are not essential for lambda replication. The mutations have been mapped between thr and leu loci in the vicinity of mutations groPC756 (dnaK gene) and groPC259 (dnaJ gene), their order being: thr-gpr2-groPC756-groPC259-gpr27. Complementation analysis using transducing phages suggested that mutations gpr2, 27 are localized in genes unknown earlier, outside dnaK and dnaJ genes.     

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.     

An intermediate in the phage lambda site-specific recombination reaction is revealed by phosphorothioate substitution in DNA.

It has been proposed that phage lambda site-specific recombination proceeds via two independent strand exchanges: the first exchange forming a Holliday-structure which is then converted into complete recombinant products by the second strand exchange. If this hypothesis is correct, one should be able to trap the putative Holliday intermediate by preventing the second strand exchange. In this paper, we show that substitution of phosphorothioate for phosphate in one strand of a recombination site is an effective way to block recombination while permitting the accumulation of a novel structure. This effect is seen only when phosphorothioate is positioned at a point of potential cleavage by Int recombinase, demonstrating that the inhibition of strand exchange is highly specific. Analysis of the novel structure that accumulates in these reactions proves that it contains a Holliday joint. Holliday-structures can also be detected in unblocked recombinations but are present at very low levels. The characteristics of Holliday-structure formation that we describe substantiate the proposed recombination pathway.     

Cloning of the replication gene P of bacteriophage lambda: Effects of increased P-protein synthesis on cellular and phage DNA replication

Summary A restriction fragment of DNA carrying the P gene was cloned in the high copy number plasmid RSF2124. Cells harbouring this new plasmid RSF2124/E complement Pam80 phage. A lac promoter-operator region (lacP), produced by EcoRI digestion of plasmid pKB252, was inserted into RSF2124/glE such that induction of the lac promoter by IPTG or lactose leads to increased production of the P gene product. A high amount of P protein in E. coli cells results in a slow inhibition of bacterial DNA synthesis, suggesting that the initiation reaction is blocked by P protein. Synthesis of DNA proceeds normally under these conditions.Nonsuppressing groPA15 mutant bacteria which are unable to support the replication of wild-type (wt), acquire the ability to replicate Pam80 phage but not wt when they are transformed with a plasmid carrying the P gene. When harbouring a plasmid containing the mutant Pamber 80 gene, groPA15 mutants are able to support the replication of wt phage when infected at a high multiplicity. Pam80 phage does not multiply in these cells.     

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.