Chi
Year: 2005
Type: article
Abstract: Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove WHEN we stumbled over Chi in coliphage λ (in 1972?), it appeared to be a uniquely accessible example of a “recombination initiator,” whose existence was implied by gene-conversion gradients (polarons) of fungi. Hence, it promised to have wide significance for our understanding of meiotic as well as of prokaryotic recombination. For a time, Chi seemed to fulfill its promise, but things turned out otherwise. Nevertheless, Chi did elucidate basic aspects of genetic recombination and genome maintenance, played a role in the development of λ as a cloning vehicle, and continues to bring enzymological surprises. In this Perspectives, which reflects my rather personal memory of events, literature citations of work from our lab are omitted to improve readability. They can be found in older reviews (e.g., Myers and Stahl 1994; Smith 1998) or electronically. For those in a hurry, here is the bottom line: Escherichia coli's RecBCD enzyme enters duplex DNA at a double-strand break and travels in a destructive mode until it encounters a properly oriented octamer called Chi. This encounter civilizes the enzyme, which keeps on traveling, in a recombinagenic mode, recruiting E. coli's strand-invasion protein, RecA, to effect recombination when a homolog is available. The primary adaptive significance of Chi is likely to concern E. coli DNA replication, when breaks occur at the fork. Since these breaks are repaired by a RecBCD-promoted recombination-like reaction (usually between the two tines of the fork), Chi plays a role in the maintenance of the E. coli genome. In 1969–1970, Mary Morgan Stahl (Figure 1) Mary Morgan Stahl (1934–1996). Mary was the driving force in the Stahl lab throughout the Chi era. and I enjoyed a sabbatical leave with Noreen and Ken Murray in Edinburgh, where we pursued our studies on relationships between DNA replication and genetic recombination in the coliphage λ. During this period, Noreen's undergraduate honors student, David Henderson, encountered a curious phenomenon while trying to “Benzerize” the region of the λ chromosome that contained the known recombination genes (Figure 2) Map of λ showing only the features referred to in this Perspectives. att is the site on the λ chromosome that recombines with a related site on the E. coli chromosome when λ is reduced to the prophage state. The product of the int gene catalyzes that reaction. The recombination genes red and gam are described in the text. Sites of Chi arising spontaneously in λ are marked as χ. . [Seymour Benzer (1961) had created a set of overlapping deletions within the rII region of phage T4 as a device for rapidly mapping any newly arising rII mutation.] Among λ's recombination genes are gam, whose product inactivates E. coli's recombination-related nuclease RecBCD, and red, which supplies recombination enzymes more compatible with λ's life style. Among the deletions that David sought were those that were missing both red and part of gam. Phage missing red and gam had been identified previously as λpbio-transducing phage. These λpbio phage occasionally arise in lysogenic cells when λ prophage, excising itself carelessly from the E. coli chromosome, picks up the E. coli bio gene in place of its own recombination region (see Anderson 1987). Such phage can be selected for by their novel ability to make (pretty-good-sized) plaques on a P2 lysogen of E. coli (the “Spi−” phenotype), a property shown to depend on the loss of both the red and gam gene functions. To select Spi− phage that were pure deletions, instead of bio substitutions, David exploited the observation that λ particles with chromosomes shortened by deletions are relatively resistant to the destabilizing effects of the Mg++-chelating agent, EDTA. Using a phage stock that had been grown in the lytic cycle, rather than having been induced from the prophage state, also helped to avoid bio substitutions. David did obtain a set of red gam deletions but found that they all made tiny (“pin-prick”) plaques. Initial attempts to grow them to a useful titer failed. Undaunted, David kept trying to grow them and succeeded when his Darwinian exercise resulted in derivatives with pretty good plaque size. He showed that each of these variants was the result of one or another single mutation, usually distant from the red gam region, that suppressed specifically the poor growth of the red gam deletion mutants. (These “Henderson suppressors” were the as-yet-unnamed Chi mutations.) David's large-plaque variants still retained the originally selected deletion and were red gam mutants, as indicated by their Spi− phenotype. In his subsequent graduate work with Jon Weil at Vanderbilt, David studied the growth-promoting phenotype of the suppressors and noted their stimulating effect on the ability of the red gam deletion phage to synthesize DNA (Henderson and Weil 1974a,b). While Mary and I were in Edinburgh, Ken McMilin, working in Germany with Enzo Russo, demonstrated that the imposition of a total block to λDNA replication did not eliminate the ability of λ to recombine and to make infectious particles. Later, Ken realized that such a strong replication block could be used to survey the distribution of exchanges along the length of the λ chromosome, using density-labeled phage. The density of each recombinant phage particle would be determined by its inheritance of DNA from one conserved parent labeled with heavy isotopes and one carrying ordinary isotopes (Figure 3) Replication-blocked λ lytic cycle crosses conducted in the absence of DNA replication produce ample phage particles for genetic analysis. When one of the two parents is heavy labeled and the other carries ordinary isotopes, the density of each of the resulting progeny particles reveals the fraction of its DNA that has been inherited from each of the two infecting parents. Selection against terminally located ts markers (Ats and Rts) allows only crossover particles to plate. If most of the crossover particles have enjoyed but one exchange, the location of that exchange is revealed by the position of the particle in a cesium formate equilibrium density gradient. The cosegregation of the cI marker with the density label implies, for most purposes, the validity of the assumption of single exchanges. Lysates centrifuged to equilibrium in a density gradient are collected as drops emerging successively through a needle hole into 1 ml of broth. Each such sample is assayed by plating at permissive temperature for total phage and at high temperature for A+R+ recombinants. Among the recombinants, the cI marker is scored from the appearance of the plaque. Phage-carrying chromosomes that have recombined by splicing the DNA duplex across the cI gene make sectored colonies interpreted as heteroduplexes (see Stahl 1994). . He limited his experiment to the host-cell recombination system as it operates on λ chromosomes. To eliminate λ's own recombination genes in one mutational step, Ken used a bio substitution that extended from att through gam (Figure 2), knocking out all the known site-specific and generalized recombination genes. The clustering of genes by function, which characterizes the λ genome, made it plausible that any unidentified recombination genes would be deleted, as well. When Mary and I returned to Eugene, Oregon, we embarked on a parallel project, to determine the distribution of exchanges along λ's chromosome in the presence of each of the combinations of λ's known recombination genes, int, red, and gam (Figure 2). To avoid the small-plaque problem of red gam double mutants, anticipated on the basis of David's work, we used a conditional (amber or sus) mutation of gam. Since the project required that we construct numerous different pairs of parental phage, we reckoned that we should first combine our markers that monitor exchange (Ats, cI, and Rts mutations) and the mutation that blocks replication (Psus) (Figure 3) with one of David's deletions, which extended from att through int, red, and gam. Separately we would construct the seven sets of recombination mutants (int, red, gam, int red, int gam, red gam, and int red gam), screening for which is best done in the absence of the markers to be used for monitoring exchanges. Then we would UV irradiate these recombination-deficient phage “heavily” to reduce their contribution of genes to the progeny of a cross, cross the UV'd phage with the Ats cI Psus80 and Psus80 Rts parents that contained the att-gam deletion, and plate the progeny phage under conditions (low temperature on a Su+ recA mutant host) that select for the loss of the deletion. This would ensure the incorporation of the desired combinations of recombination genes while leaving the Ats, Rts, Psus80, and cI markers undisturbed. The strategy worked. Mary constructed the genotypes and grew the stocks needed for the experiments. Only then did I realize that the procedure used practically guaranteed that every one of the 14 genotypes so constructed would carry a “Henderson suppressor.” Since we had no understanding of whether or not the suppressor might alter genetic recombination, we had no choice but to rebuild all 14 phage using a different strategy (from which I will spare you). Mary quit the lab in protest of my thoughtlessness. At about this time, Ken noted that crossing over between his λpbio phage was concentrated in and near the bio substitution! This provoked the hypothesis that bio DNA carried a “Henderson suppressor” and that the suppressor was a hotspot for crossing over. However, an insight was needed to connect plaque size with recombination. The connection was made by the observations of Enquist and Skalka (1973), who noted that recombination-deficient (red gam) phage λ infecting wild-type E. coli appeared to produce predominantly circular DNA monomers rather than the more complex intracellular forms made by recombination-proficient λ. Since λ monomers (unlike dimers and higher multimers) are poor substrates for being packaged into phage particles (Szpirer and Brachet 1970), inefficient packaging could account for the small plaque size of the red gam mutant phage. The inability of these recombination-deficient mutants to produce suitable packaging precursors could, in turn, reflect the monomers' inability to recombine with each other to generate dimers as shown in Figure 4 Replication of λ in its lytic cycle. The linear DNA, injected by a phage particle into E. coli, circularizes, generating the sequence called cos. In wild-type infections, replication in the theta mode, which generates monomer circles, switches to sigma (rolling circle) replication, probably by breakage of replication forks (Enquist and Skalka 1973). Packaging of the DNA into phage heads proceeds efficiently only from dimers or multimers, which contain two (or more) cos sites. Such multimers can arise by recombination or by sigma replication. This simplified figure fails to show the interrelationship between those two processes and unrealistically diagrams the recombination as reciprocal. . As described below, we gained support for this possibility. [However, David's earlier observation that the suppressor stimulated DNA replication implied that multimer formation by recombination was not the whole explanation for the phenotype of the suppressor, and subsequent work confirmed his views: the Henderson suppressor does appear to enhance rolling circle replication (Figure 4, and see Dabert et al. 1992).] On the basis of the considerations above, we guessed that Henderson's suppressor was a cis-acting recombination initiator whose activity was manifest in the absence of λ recombination functions—and we realized that Mary's phage stocks containing the suppressor provided exactly the right material for testing that idea. We suggested to Steve Lam, undergraduate honors student, that he test the idea using the red gam mutant phage set. As expected, Steve found that the stocks did contain a Henderson suppressor (judged by plaque size). The suppressor mapped near the right end of λ's conventional linkage map, and, bingo, the phage carrying it did recombine like gangbusters in that region (Figure 5) Density-labeled, replication-blocked crosses of red gam mutant λ in the absence of Chi (left) and in the presence of Chi (χD; right). Density-labeled crosses blocked for DNA synthesis (Figure 3) reveal a low, roughly uniform rate of exchanges along the length of the chromosome in the absence of Chi. In the presence of χD, near λ's right end, the rate of exchange is elevated near the Chi, mostly in the interval to the right of the cI marker (open circles), but evidently extending to the left of that marker for about half the length of λ. (The magnitude of the increase implies that Chi civilizes RecBCD thousands of times better than does the average octamer in λ.) In the absence of Chi, sectored plaques (half-solid) were not reliably scored because of the reduced plaque size resulting from the nonconditional red mutation. Due to the poor phage yield of the cross lacking Chi, unadsorbed parental Rts phage make a conspicuous contribution in the light peak of total phage (triangles). The left panel is from Stahl et al. the right panel is from et al. . Mary was was about this time, at in that I first our Jon Weil was in and was that the phenotype of the suppressors was related to recombination hotspot However, he our to and David confirmed our the or our two made the suppressors that were at They showing a between recombination and the of suppressor In our this these it was that the suppressors in cis-acting of recombination, and we them first and then Chi hotspot with the being used by Henderson and Weil and by As suggested by the crosses with λpbio phage, described above, Chi is in E. coli This was by that phage induced from other on the chromosome had a phenotype like that of red gam mutant derivatives of λ phage into which of DNA had been at plaque size and a high recombination rate near the of Chi, about half of these the that was about one of E. coli This was confirmed by the genome The of λ that carried of revealed that Chi was in DNA at a and could in red gam mutant λ However, by both and to reveal any activity for Chi in or meiotic more that Chi could in a cross between λ prophage carried out by Since most prophage genes are this result made it that Chi activity was to λ. Then that demonstrated Chi activity in the E. coli gene in the total absence of λ. both Chi recombination when it was in the or the DNA, about the of Chi The of mutant provided by the that Chi was only in the wild-type recombination of E. coli, at that referred to as the The of this is RecBCD, a nuclease on linear DNA see and Since the inactivates RecBCD, and the λ with an this implies that Chi in λ could have been only in a red gam double was a cycle of and experiments. with revealed the Chi, in an ordinary red gam mutant stimulated recombination only to its left the conventional λ linkage The extended half the length of (Figure “recombination a of Chi. Chi when in both or when in only one of the two parents in the phage cross Chi stimulated recombination when it was in a carried by only one of the two parents in the recombination was to the region of to the left of the substitution and was conspicuous when that was distant from the Chi against my showed when such a substitution was the Chi was was the first of genetic conducted in our and helped show that the of Chi activity of where in the λ chromosome the Chi was out across Chi as the of Chi and with DNA or DNA Due to the of the end site the site whose the of the λ chromosome (Figure each of these processes is with to the λ who had his by recombination and packaging of λDNA in then and showed that the Chi was by of cos. Chi then to its On this Chi was to be a and, in a of DNA was shown to be the see Smith et al. Mary to show that a Chi can also be by a double-strand break in by a enzyme from the Chi. This provided strong support for the that cos can Chi because cos is a double-strand break an The in of the RecBCD enzyme that known as with a understanding of the of provided of about the In the enzyme could be under the to linear duplex DNA at an The enzyme through the or not the DNA, on the in Smith a cos or any other double-strand break could be an for the enzyme, with cos being the one in λ. on λ packaging revealed that the enzyme that cos in for λDNA at the left end of the λ chromosome et al. This could account for the for of Chi, with to for Chi to one had only to that blocked the of RecBCD into the left end of a chromosome about to The for Chi to cos implied that an enzyme from cos to Chi the sequence from the as to to the Chi. the E. coli chromosome also have for from and Mary's that replication of λ the activity of a Chi carried by the λ. This to a in which replication forks to were for RecBCD This work, with Henderson's observation of DNA replication, implied of DNA replication and genetic recombination by Skalka and to the that the role of E. coli's RecBCD recombination for this and for an role for Chi in the from the E. coli genome project, which revealed that the Chi on of the replication to be each in the that would RecBCD to to them in its of in conditions that a traveling, RecBCD a in the DNA at Chi et al. In the for recombination that was at the and such a could as the and the a of on that account (e.g., Smith and Stahl for a testing its and the in the that resulted from the of observation and The was The of that out the previously of the RecBCD enzyme of about Chi et al. and David and noted in a mutant a red gam λ cross as were a Chi sequence at the right end of the λ chromosome (the site for Among David's was the that the role of Chi is to activity from the RecBCD This would the enzyme from a into one to recombination see et al. with these the of whether or not recombination was both made in a single of λ crosses had indicated that was as by the of phage particles. However, that the of the of packaging from a by recombination. he crosses that packaging from the cos that of he found that the of was For we that to that the recombination stimulated by Chi in a we a in on a of from The by that which described the of recombination, and we were personal as well as with of was he that of our about recombination were In it was of to that recombination could be in a He out that this enzyme linear DNA in property of the enzyme that we under the and that the role of Chi be to the is no he that the two could generate both crossover when the DNA to the right of Chi on one of the λ had been In so as we he it that a duplex into the We to the test in crosses that the of of the two infecting phage. When the phage was in we when the parent was in the the recombinant that would have inherited DNA to the right of Chi from that parent was relatively all as by the of a reaction. the of in the and Mary to to our lab to our by to within the He that I should to of other could he have within that rapidly system was an but that is another When I did to I was to a graduate student, where I had to an However, to be as as his the end of the was that he should to He did and in the of the Chi in During his in Eugene, oriented our Chi the role of Chi and RecBCD in the E. coli chromosome of the literature with his to a of (e.g., Myers and up for an set of the that Chi civilizes RecBCD by or the They red gam mutant λ crosses in cells with accessible Chi on a that could be at a cos The λ phage recombined as they were so in a mutant When such crosses were conducted in cells that protein, the crosses like ordinary red gam mutant the RecBCD has been by a cis-acting Chi, where the exchange will that with a set of λ When Chi, in only one of two λ is a the ability of Chi to the is by the of the two parental phage. When at Chi is to a high of the the exchanges occur between chromosomes. When at Chi is to the of that recombination between the and parents with force the of the a RecBCD enzyme keeps on until it and effects recombination. The of this is to in in other (e.g., and and Smith which are to a from the of the RecBCD et al. has been that the destructive activity of RecBCD is E. coli's against DNA and that Chi is E. coli's against of In a it be an of the If wild-type λ contained one every in E. Chi might still be of my my and, along with provided The E. was throughout the of this with their and their
Source: Genetics
Author: Franklin W Stahl
Institution: University of Oregon
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2137607679