Proc. Nat. Acad. Sci. USA Vol. 72, No. 6, pp. 2188-2192, June 1975
Excision of Prophage X in a Cell-Free System (site-specific recombination/transfection)
SUSAN GOTTESMAN* AND MAX GOTTESMAN Section on Biochemical Genetics, Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
Communicated by A. D. Kaiser, March 21, 1976 A cell-free system that promotes the exABSTRACT cision of prophage X DNA has been established. The substrate for the reaction is phage DNA carrying two attachment sites, which, in vivo, undergoes intramolecular recombination between these sites. The in vitro recombination system is efficient; 25-35% of the substrate DNA undergoes recombination in 30 min. There is an absolute requirement for ATP; Mg++ and spermidine are stimulatory. RNA does not appear to be involved, nor can a role for DNA synthesis be demonstrated.
The DNA of prophage X is inserted covalently in the DNA of its bacterial host, Escherichia coli. Excision of the prophage DNA follows expression of two phage-specified proteins, Int and Xis. These functions promote recombination between the prophage-bacterial DNA junctions (attL and attR). Int and Xis are site-specific; they do not stimulate recombination between homologous DNA molecules, nor do they promote the excision of prophage DNA with a different insertion site from X. As yet, no host function has been implicated in excisional recombination (see review by Gottesman and Weisberg, ref. 1). The excision reaction is extremely efficient; virtually all of the prophage DNA excises after induction. In addition, the reaction occurs with inert DNA molecules; i.e., inhibitors of protein (chloramphenicol) RNA (rifampicin), or DNA (nalidixic acid) synthesis appear not to inhibit the excision process itself (3). These features, the efficiency and simplicity of excisional recombination, recommend it for study in vitro. We report here a recombination system in vitro in which excision of X prophage DNA occurs at high frequency and with properties similar to those seen in vivo. RESULTS Assay for Prophage Excision In Vitro. The substrate for excisional recombination in vitro is the DNA of the X variant, Xatt2. The choice of this substrate is based on two considerations: (1) Xatt2 DNA carries the two attachment sites that normally bracket an integrated prophage, attL and attR (Fig. 1). In vivo, Xatt2 DNA undergoes intramolecular recombination between attL and attR. This self-excision is promoted by Int and Xis, and is extremely efficient, even more efficient than intermolecular recombination between these att loci (2) (see below). Furthermore, an intramolecular reaction follows first-order rather than second-order kinetics. This might permit the use of lower DNA substrate concentrations. * Current address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
(2) The products of the intramolecular recombination of Xatt2 DNA are a molecule of 27 X 106 daltons (here referred to as X DNA) and the fragment of bacterial DNA bracketed by attL and attR (Fig. 1). Both Xatt2 DNA and X DNA are equally capable of infecting spheroplasts (with an efficiency of 10-i phage formed per phage X equivalent) (3); the former yields Xatt2 phage, and the latter X phage. The two phage can be readily distinguished by their sensitivity to chelating agents such as EDTA or pyrophosphate. Xatt2 phage, with a DNA content of 34 X 106 daltons, is exquisitely sensitive to these agents; X phage, with its reduced genome, is completely resistant. The excision reaction can therefore be assayed thus: (a) Xatt2 DNA is incubated with an E. coli extract. (b) The DNA is isolated and mixed with spheroplasts. (c) After incubation, the spheroplasts release phage, which are titered in the presence and absence of chelating agents. Since the spheroplasts are incapable of supporting prophage excision, the frequency of resistant phage is a measure of recombination activity in vitro. The spheroplast system has already been tested as an assay for recombination in vivo (3). DNA Substrate for the Excision Reaction. We have tested the relative effectiveness of linear (as extracted from phage) Xatt2 DNA, closed covalent Xatt2 circles, and (as suggested by Nash) (13) Hershey circles (formed by annealing the sticky ends of linear Xatt2) as substrates for site-specific excision in vitro. The data in Table 1 show that both Hershey circles and closed covalent circles are excised equally efficiently. Linear DNA, on the other hand, is converted to a form ineffective in producing phage from spheroplasts. Sucrose gradient analysis (not shown) of the linear DNA after incubation with the in vitro mixture indicates that the DNA remains at Xatt2 size, suggesting that the sticky ends of the molecules are inactivated, but that no extensive degradation of the double-stranded portion of the molecule occurs. The remaining active DNA does not appear to excise, but too few molecules remain to conclude that the excision reaction itself requires DNA circularizing. Genetic Requirements for the Excision Reaction. In Exp. I (Table 2), cells lysogenic for the thermoinducible phage XcI857 were transiently induced (15 min at 420), superinfected with Xatt2int2xisl, and then incubated an additional 90 min at 300. DNA was extracted from these cells and added to spheroplasts. Note that excisional recombination, as measured by the appearance of pyrophosphate-resistant phage in the spheroplast lysate, depends in vivo upon the presence of both Int and Xis in the induced lysogen. These functions must be present in the same cell; no complementa2188
Proc. Nat. Acad. Sci. USA 72
(1975)
X Excision in a Cell-Free
TABLE 1. Site-specific excision of various DNAs in vitro gal
DNA Linear Hershey circles Closed covalent circles
Infectious DNA remaining (%) 2 40 51
Excision (%)
Excision of Prophage X in a Cell-Free System (site-specific recombination/transfection)
SUSAN GOTTESMAN* AND MAX GOTTESMAN Section on Biochemical Genetics, Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014
Communicated by A. D. Kaiser, March 21, 1976 A cell-free system that promotes the exABSTRACT cision of prophage X DNA has been established. The substrate for the reaction is phage DNA carrying two attachment sites, which, in vivo, undergoes intramolecular recombination between these sites. The in vitro recombination system is efficient; 25-35% of the substrate DNA undergoes recombination in 30 min. There is an absolute requirement for ATP; Mg++ and spermidine are stimulatory. RNA does not appear to be involved, nor can a role for DNA synthesis be demonstrated.
The DNA of prophage X is inserted covalently in the DNA of its bacterial host, Escherichia coli. Excision of the prophage DNA follows expression of two phage-specified proteins, Int and Xis. These functions promote recombination between the prophage-bacterial DNA junctions (attL and attR). Int and Xis are site-specific; they do not stimulate recombination between homologous DNA molecules, nor do they promote the excision of prophage DNA with a different insertion site from X. As yet, no host function has been implicated in excisional recombination (see review by Gottesman and Weisberg, ref. 1). The excision reaction is extremely efficient; virtually all of the prophage DNA excises after induction. In addition, the reaction occurs with inert DNA molecules; i.e., inhibitors of protein (chloramphenicol) RNA (rifampicin), or DNA (nalidixic acid) synthesis appear not to inhibit the excision process itself (3). These features, the efficiency and simplicity of excisional recombination, recommend it for study in vitro. We report here a recombination system in vitro in which excision of X prophage DNA occurs at high frequency and with properties similar to those seen in vivo. RESULTS Assay for Prophage Excision In Vitro. The substrate for excisional recombination in vitro is the DNA of the X variant, Xatt2. The choice of this substrate is based on two considerations: (1) Xatt2 DNA carries the two attachment sites that normally bracket an integrated prophage, attL and attR (Fig. 1). In vivo, Xatt2 DNA undergoes intramolecular recombination between attL and attR. This self-excision is promoted by Int and Xis, and is extremely efficient, even more efficient than intermolecular recombination between these att loci (2) (see below). Furthermore, an intramolecular reaction follows first-order rather than second-order kinetics. This might permit the use of lower DNA substrate concentrations. * Current address: Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
(2) The products of the intramolecular recombination of Xatt2 DNA are a molecule of 27 X 106 daltons (here referred to as X DNA) and the fragment of bacterial DNA bracketed by attL and attR (Fig. 1). Both Xatt2 DNA and X DNA are equally capable of infecting spheroplasts (with an efficiency of 10-i phage formed per phage X equivalent) (3); the former yields Xatt2 phage, and the latter X phage. The two phage can be readily distinguished by their sensitivity to chelating agents such as EDTA or pyrophosphate. Xatt2 phage, with a DNA content of 34 X 106 daltons, is exquisitely sensitive to these agents; X phage, with its reduced genome, is completely resistant. The excision reaction can therefore be assayed thus: (a) Xatt2 DNA is incubated with an E. coli extract. (b) The DNA is isolated and mixed with spheroplasts. (c) After incubation, the spheroplasts release phage, which are titered in the presence and absence of chelating agents. Since the spheroplasts are incapable of supporting prophage excision, the frequency of resistant phage is a measure of recombination activity in vitro. The spheroplast system has already been tested as an assay for recombination in vivo (3). DNA Substrate for the Excision Reaction. We have tested the relative effectiveness of linear (as extracted from phage) Xatt2 DNA, closed covalent Xatt2 circles, and (as suggested by Nash) (13) Hershey circles (formed by annealing the sticky ends of linear Xatt2) as substrates for site-specific excision in vitro. The data in Table 1 show that both Hershey circles and closed covalent circles are excised equally efficiently. Linear DNA, on the other hand, is converted to a form ineffective in producing phage from spheroplasts. Sucrose gradient analysis (not shown) of the linear DNA after incubation with the in vitro mixture indicates that the DNA remains at Xatt2 size, suggesting that the sticky ends of the molecules are inactivated, but that no extensive degradation of the double-stranded portion of the molecule occurs. The remaining active DNA does not appear to excise, but too few molecules remain to conclude that the excision reaction itself requires DNA circularizing. Genetic Requirements for the Excision Reaction. In Exp. I (Table 2), cells lysogenic for the thermoinducible phage XcI857 were transiently induced (15 min at 420), superinfected with Xatt2int2xisl, and then incubated an additional 90 min at 300. DNA was extracted from these cells and added to spheroplasts. Note that excisional recombination, as measured by the appearance of pyrophosphate-resistant phage in the spheroplast lysate, depends in vivo upon the presence of both Int and Xis in the induced lysogen. These functions must be present in the same cell; no complementa2188
Proc. Nat. Acad. Sci. USA 72
(1975)
X Excision in a Cell-Free
TABLE 1. Site-specific excision of various DNAs in vitro gal
DNA Linear Hershey circles Closed covalent circles
Infectious DNA remaining (%) 2 40 51
Excision (%)
