Mary M. Stahl

Rec-mediated recombinational hot spot activity in bacteriophage lambda: III. Chi mutations are site-mutations stimulating Rec-mediated recombination☆☆☆

Lambda phage defective for Red and gam function make small plaques on rec+ bacteria. Mutants (called Chi) of λ arise which suppress the small-plaque phenotype. Chi mutations arise at at least four well-separated sites; one site is between gene L and att, one is between att and gam, one is in the cII gene, and one is near gene S. A phage strain carrying a Chi mutation at a given site has an extraordinarily high rate of Rec-mediated crossing-over near that site.

A role for recombination in the production of “free-loader” lambda bacteriophage particles

Abstract Density-labeled crosses were performed with bacteriophage lambda under conditions which diminish DNA duplication. The production of viable phage containing fully conserved parental DNA was found to be dependent upon the action of the genetic recombination systems. The production of phage containing DNA with one newly synthesized chain was less dependent upon recombination. The production of phage with chromosomes both of whose chains were synthesized following infection show little, if any, dependence on recombination. One can speculate that some step in the maturation process of bacteriophage lambda is inseparable from the reduction of lambda DNA to the monomeric rods characteristic of lambda virions.

Improved in vitro packaging of coliphage lambda DNA: a one-strain system free from endogenous phage

In previous systems for in vitro packaging of lambda DNA, phages are produced from the packaging components as well as from added DNA. We have developed a new genetic strategy for in vitro packaging that bypasses this endogenous phage problem. Our system employs a single bacterial strain whose lambda prophage codes for all of the packaging proteins but is deleted for cos, the packaging origin. Crude extracts of the single lysogen: (i) are virtually free from endogenous phages, (ii) package added lambda DNA efficiently and (iii) are easy to prepare. Using the cos- in vitro packaging system we show that packaging of lambda linear monomers is a second-order reaction, but that packaging from concatemers prepared by annealing or ligation is first order. We conclude that in our cos- system, linear monomers are a poor substrate for in vitro packaging but that packaging from concatemers works well.

Rec-mediated recombinational hot spot activity in bacteriophage λ

SummaryA Chi mutation in phage λ stimulates Rec-mediated crossing over more to one side of itself than to the other; stimulation, which is maximal near Chi, can occur at some distance from the Chi site as well. A gross heterology differentiating the two recombining parents does not interfere with the distant Chi-stimulated crossover whether the heterology is at the Chi site or between the Chi site and the distant interval in which recombination is monitored. These conclusions hold whether recombination is measured “genetically” in standard crosses or “physically” in density-labeled crosses conducted in the absence of DNA replication.

Orientation-dependent recombination hotspot activity in bacteriophage λ☆

Promoters of genetic exchange by the Escherichia coli Rec system, Chi elements, have been analyzed in λ phages carrying bacterial EcoRI restriction fragments. Some fragments confer Chi+ phenotype in one orientation and Chi− in the opposite orientation. The inactivity of Chi in one orientation explains why all active Chi elements in λ manifest a certain recombinational bias of the same sense. When these studies were undertaken, we rather expected to find two classes of Chi, one class which stimulated recombinant formation stronger to its left and one class stimulating recombinant formation more strongly to its right. The failure to find the second class is now understandable by supposing that the orientation of Chi which would have permitted it to act rightward is the orientation in which Chi has no activity at all. Several models are proposed for the orientation dependence of Chi activity.

Double-chain-cut sites are recombination hotspots in the red pathway of phage λ

The Red recombination pathway of phage lambda is shown to target recombination to double-chain ends of DNA. A double-chain cut, delivered in vivo to only one of two parents participating in a lambda lytic cross by a type II restriction endonuclease, increases the proportion of crossing over in the interval containing the cut compared with other intervals. The stimulating effect of a cut is evident whether replication is inhibited or permitted. Cut stimulation can move away from the initial cut-site, presumably by double-chain degradation. Movement of the stimulating effect of a cut is dependent on the Escherichia coli gene recA when the cross is carried out under conditions that inhibit phage replication. When replication is permitted, all aspects of cut-stimulated recombination are independent of recA. Evidence is presented to show that the reaction that is stimulated by cutting is often non-reciprocal at the molecular level.

In phage λ, cos is a recombinator in the red pathway☆

Among λ particles carrying chromosomes that have failed to replicate during a lytic cycle cross there is a high frequency of Red-mediated recombination near the right-hand end. Earlier work has shown that this recombination is dependent on cos (cohesive end site), the packaging origin of λ. In contrast to the prediction of the break-copy model proposed earlier, we find a high recombination rate near cos even when only one of the two participating parents has a functional cos at that locus. The exchange is accompanied by loss of the stimulating cos in the recombination product, irrespective of the marker configurations: a+b+cos− rather than a+b+cos+ is produced in the cross a+b−cos− × a−b+cos+ as well as in the cross a+b−cos+ × a−b+cos−. Further analyses of these and earlier data allow the formulation of a detailed model for cos-stimulated, Red-mediated genetic exchange. In this model, cos stimulates exchange by virtue of being a double-strand cut site. The model has several features like that proposed for yeast. This role of cos in the Red pathway contrasts with the role of cos in the RecBC pathway, in which cos serves as an entry site for a recombinase that stimulates exchanges far from cos.

Hotspots for generalized recombination in the Escherichia coli chromosome

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA+ function, the site-specific stimulation of recombination required recB+ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology. The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 103 bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.

Evidence that the normal route of replication-allowed Red-mediated recombination involves double-chain ends.

Recombination mediated by the Red pathway of bacteriophage lambda is focused towards sites of double‐chain cuts. Double‐chain ends created either by type II restriction enzymes acting at unmodified recognition sites or by lambdas packaging enzyme, terminase, acting at cos are utilized in a manner similar to the double‐chain break repair pathway of recombination in yeast. When lambda is allowed to recombine during replicative growth, spontaneous recombination is approximately evenly distributed along the chromosome. It has been proposed that replication‐allowed recombination also is initiated by double‐chain ends. In order to test this hypothesis we ask if the in vivo expression of the Mu gam protein is inhibitory to Red recombination. Mu gam has been shown in vitro to bind to linearized duplex DNA and to shield bound DNA from exonucleases. The expression of Mu gam is found to be inhibitory to Red recombination whether replication is blocked or allowed. As a control we ask if Mu gam inhibits Int‐mediated recombination. It has been well documented that the Int pathway of recombination does not involve any double‐chain breaks and, consistent with this, the Int pathway is not inhibited by Mu gam. We suggest that the in vivo expression of Mu gam or other similar activities may be a generally useful way to determine if those processes that respond to an artificially introduced double‐chain cut normally involve double‐chain ends.

Red-mediated recombination of phage lambda in a recA− recB−host

SummaryThe lambda Red recombination system works poorly among unreplicated gam+ lambda chromosomes in recA- cells compared to recA+ cells. Recombination is not enhanced in recA- recB-cells. Thus, the inability of Red to promote recombination in recA- replication-blocked cross is not due to the hypothetical destruction of recombination intermediates by the recB nuclease. This conclusion strengthens previous proposals that the products of the red genes can operate upon recombinational intermediates which require recA activity for their formation.