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Lambda site-specific recombination: The att site
Cell, Vol. 25. 585-586.
September
1981,
Copyright
0 1981
by MIT
Lambda Site-Specific Recombination: The aft Site Susan Gottesman Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20205
The temperate bacterial virus X integrates its DNA into the host chromosome during the establishment of lysogeny. The integration reaction depends on phage protein Int and bacterial proteins; the excision reaction, part of the bacteriophage induction pathway, requires an additional phage-coded protein, Xis. Both integration and excision can be carried out efficiently in vivo and in vitro, and the extensive genetics of the h system has led to rapid progress in the characterization of this site-specific recombination reaction. It is specific for both donor and recipient sites (termed att, for attachment site); by its efficiency; and by its reciprocity, in that both products can be recovered in good yield from a single reaction. We will concentrate here on what has been learned about the site of the recombination itself and about the phage and bacterial attachment sites, and on what these structures tell us about the nature of the recombination event. Two aspects of attachment-site structure, hinted at by early genetic experiments, have recently been confirmed by biochemistry and DNA sequencing studies. First, it was known that the phage and bacterial attachment sites differ in that particular pairs of sites recombine at very different efficiencies. A nomenclature to indicate these differences postulates the presence of a crossover region, common to both phage and bacterial att sites, called 0. In addition, each site was postulated to have unique flanking arms to this 0 core, called B and B’ for the bacterial site and P and P’ for the phage site. Thus integration (phage site POP’ x bacterial site BOB’) is an efficient reaction, while other reactions (BOB’ X BOB’, for instance) are not efficiently carried out by the Int system. Landy and Ross (Science 797, 1147-l 160, 1977) settled the question of homology between the phage and bacterial attachment sites with the sequence of both phage and bacterial art sites, showing the existence of an AT-rich common core of 15 bp (see figure for sequence). Given the finding of a common core of 15 bp, and the earlier observation that not all attachment sites are equal, the attachment sites must consist of more than the core alone. Hsu et al. (Nature 285, 85-91, 1980) and Mizuuchi and Mizuuchi (PNAS 77, 32203224, 1980) defined the limits of structure needed for site-specific recombination by gradually reducing the size of cloned att sites and testing the resulting plasmids for their ability to undergo site-specific recombination in vitro with cloned, intact att POP’ and art BOB’. Their results indicate that the phage aft site loses its ability to act as an efficient att POP’ if it has less than 152 bases of the P arm and less than 68
bases of the P’ arm. The BOB’ site, however, requires only the 15 bp core for efficient in vivo recombination; in vitro, about 4 more bases on each side of the core are necessary for efficient recombination (Mizuuchi et al., CSHS, in press). In fact, an art POP’ with less than 56 bases of the P’ arm and 73 bases of the P arm not only loses its ability to act as POP’, but acquires the ability to act as BOB’. Thus BOB’ can be defined by the 15 bp core, possibly a few bases on each side, and lack of arm information necessary for POP’. What function do these structures have in the recombination reaction? The role of the core for BOB’ can be tested with variants of the bacterial attachment site. E. coli has been kind enough to donate these variant attachment-site sequences to the cause of lambdologists; Shimada et al. (JMB 63, 483-503, 1972) observed that if the normal bacterial attachment site aft BOB’ is deleted, lambda will continue to integrate, at 0.5% the normal frequency, at a variety of “secondary attachment sites.” Phages can be induced from these sites; some will excise abnormally and will carry adjacent bacterial DNA rather than reforming the phage attachment site. Sequencing of the attachment-site region from these abnormal excision events yields two kinds of information. First, the bacterial sites with which POP’ recombines contain sequences resembling the core. A survey of the most conserved bases indicates that the TTTTTTT stretch may play a particularly critical role. Second, the crossover region can be defined by the site of sequence divergence from the correct core as one moves from the phage into the bacterial end of the recombinant. The figure shows this for one of the most frequently used secondary sites, that in the proAB region (Pinkham et al., JMB 744, 587-592, 1980). Correlation of a set of such recombinants indicates that the transition may occur from the third T of the core, reading from the left, to about 7 bases farther to the right (Mizuuchi et al., op. cit.). Mizuuchi et al. (op. cit.) have recently carried out an elegant experiment to test directly for the site of crossover in the site-specific integration reaction. In vitro recombination was carried out between unlabeled att POP’ DNA and aft BOB’ DNA carrying one strand labeled with 32P adjacent to one of the 4 bases. After recombination, the labeled BOP’ and POB’ fragments were digested with restriction enzymes, purified and stored to allow accumulation of strand breaks from “P disintegration. Analysis of the fragments generated by the 32P cleavages on an acrylamide sequencing gel provided a series of bands radioactively labeled in the bacterial portion. For instance, for the BOP’ attachment site, the pieces seen as radioactive bands would carry all of P’ (unlabeled) and the portion of labeled B to the right of the 32P disintegration. The P’ piece formed by the disintegration of the 32P closest to the crossover site would carry no B material, and therefore no radioactive label, and would not be seen
Cell 586
at
BOB’
on the gel. By determining the sequence of this missing piece for a series of recombinations using, in turn, each deoxyribonucleotide labeled with 32P, and each strand separately labeled, one could determine the point in the sequence where bacterial information ends and phage att site information begins. The results show that there are two unique crossover points, one on each strand, located as indicated in the figure, resulting in a 5’ overlap. Because the phosphorus atoms rather than the bases were labeled, the results are consistent with either a 5 bp or 7 bp separation between the two crossover points. Only the 7 bp result, however, is consistent with the results obtained from analysis of secondary-site recombination events. Studies on the properties of mutations in the core of att POP’ (called saf, for site-affinity mutations) have helped to define the roles of particular bases in POP’ and BOB’ and the role of homology between the cores in promoting efficient recombination. Weisberg (Mizuuchi et al., op. cit.) obtained such mutants among phages excised from an abnormal attachment site in galT. Phages carrying three changes in the crossover region recombine at one twentieth of normal efficiency with att BOB’; this decrease may reflect changes both in the Int recognition sequences and in homology between the cores. Phages carrying these same three changes recombine tenfold better with the abnormal ga/T att site (from which they were derived, and with which they share 11 of 15 bp of homology in the core) than do wild-type phages (with 8 of 15 bases of the core homologous). If these phage core sequences are important for Int recognition, the positive contribution of homology may be even more important than suggested by this tenfold improvement. It is clear, however, that base homology in the core is not sufficient for site-specific recombination to proceed efficiently; Shulman and Gottesman (op. cit.) recombined BOP’ carrying the core mutation att24 (missing one T in the run of six Ts in the core) with POB’ also carrying att24, and found that the mutant x mutant recombination did not proceed any more efficiently than mutant X wild-type recombination. The core therefore seems to provide both a homologous region for crossovers and essential recognition sequences for the proteins involved in the site-specific recombination reaction.
One can determine the precise location of recognition regions within POP’ and BOB’ sites by determining the protection of these regions from pancreatic DNAase and neocarzinostatin by the proteins involved in the recombination. Int, the first to be purified of the recombination proteins, protects a central core of about 30-35 bases disposed symmetrically around the core in POP’. Only a subset of these bases, including the left-hand portion of the common core, is protected in BOB’. In addition, Int apparently binds to and protects three other sites in POP’: two small sites (15-20 bp) in the P arm, and one larger site (30-35 bp) in the P’ arm (Ross et al., Cell 78, 297-307, 1979; Hsu et al., op. cit.). The P’ arm shows particularly tight binding; only the binding to this site is heparin-resistant. No homology has been seen between the sequences protected in the arms and the core-binding sequence, but some homology can be seen between sites in P and a sequence in the P’protected region. This distinction between core sites and arm sites may suggest that these two different sets of sequences respond to different Int domains (Hsu et al., op. cit.). Since one of the P binding sites and much of the P’ region fall within the limits assigned for POP’ by the DNA “nibbling” experiments described above, these sites may represent the necessary limits of the attachment site. Loss of these protected regions (with retention of the POP’ core region) are sufficient to make POP’ recombine like BOB’; the arm sites therefore must play an essential part in the POP’-BOB’ discrimination by the Int system. The phage X has assigned two gene products, 240 bases of essential site and a variety of regulatory circuits to the job of ensuring efficient integration of its DNA into the host on lysogeny and rapid escape on induction. The crossover event, a staggered cut with a homologous core, leaves both parents intact after a cycle of integration and excision. Current work in a number of laboratories on the interaction of other recombination proteins (host factors and Xis) with the aft sites, as well as studies of mutations in both the core and the arm sites, should fill in the details of this outline. I would like to acknowledge the help of Bob Weisberg. Max Gottesman and Michael Gottesman in commenting uscript.
Howard Nash, on this man-
September
1981,
Copyright
0 1981
by MIT
Lambda Site-Specific Recombination: The aft Site Susan Gottesman Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20205
The temperate bacterial virus X integrates its DNA into the host chromosome during the establishment of lysogeny. The integration reaction depends on phage protein Int and bacterial proteins; the excision reaction, part of the bacteriophage induction pathway, requires an additional phage-coded protein, Xis. Both integration and excision can be carried out efficiently in vivo and in vitro, and the extensive genetics of the h system has led to rapid progress in the characterization of this site-specific recombination reaction. It is specific for both donor and recipient sites (termed att, for attachment site); by its efficiency; and by its reciprocity, in that both products can be recovered in good yield from a single reaction. We will concentrate here on what has been learned about the site of the recombination itself and about the phage and bacterial attachment sites, and on what these structures tell us about the nature of the recombination event. Two aspects of attachment-site structure, hinted at by early genetic experiments, have recently been confirmed by biochemistry and DNA sequencing studies. First, it was known that the phage and bacterial attachment sites differ in that particular pairs of sites recombine at very different efficiencies. A nomenclature to indicate these differences postulates the presence of a crossover region, common to both phage and bacterial att sites, called 0. In addition, each site was postulated to have unique flanking arms to this 0 core, called B and B’ for the bacterial site and P and P’ for the phage site. Thus integration (phage site POP’ x bacterial site BOB’) is an efficient reaction, while other reactions (BOB’ X BOB’, for instance) are not efficiently carried out by the Int system. Landy and Ross (Science 797, 1147-l 160, 1977) settled the question of homology between the phage and bacterial attachment sites with the sequence of both phage and bacterial art sites, showing the existence of an AT-rich common core of 15 bp (see figure for sequence). Given the finding of a common core of 15 bp, and the earlier observation that not all attachment sites are equal, the attachment sites must consist of more than the core alone. Hsu et al. (Nature 285, 85-91, 1980) and Mizuuchi and Mizuuchi (PNAS 77, 32203224, 1980) defined the limits of structure needed for site-specific recombination by gradually reducing the size of cloned att sites and testing the resulting plasmids for their ability to undergo site-specific recombination in vitro with cloned, intact att POP’ and art BOB’. Their results indicate that the phage aft site loses its ability to act as an efficient att POP’ if it has less than 152 bases of the P arm and less than 68
bases of the P’ arm. The BOB’ site, however, requires only the 15 bp core for efficient in vivo recombination; in vitro, about 4 more bases on each side of the core are necessary for efficient recombination (Mizuuchi et al., CSHS, in press). In fact, an art POP’ with less than 56 bases of the P’ arm and 73 bases of the P arm not only loses its ability to act as POP’, but acquires the ability to act as BOB’. Thus BOB’ can be defined by the 15 bp core, possibly a few bases on each side, and lack of arm information necessary for POP’. What function do these structures have in the recombination reaction? The role of the core for BOB’ can be tested with variants of the bacterial attachment site. E. coli has been kind enough to donate these variant attachment-site sequences to the cause of lambdologists; Shimada et al. (JMB 63, 483-503, 1972) observed that if the normal bacterial attachment site aft BOB’ is deleted, lambda will continue to integrate, at 0.5% the normal frequency, at a variety of “secondary attachment sites.” Phages can be induced from these sites; some will excise abnormally and will carry adjacent bacterial DNA rather than reforming the phage attachment site. Sequencing of the attachment-site region from these abnormal excision events yields two kinds of information. First, the bacterial sites with which POP’ recombines contain sequences resembling the core. A survey of the most conserved bases indicates that the TTTTTTT stretch may play a particularly critical role. Second, the crossover region can be defined by the site of sequence divergence from the correct core as one moves from the phage into the bacterial end of the recombinant. The figure shows this for one of the most frequently used secondary sites, that in the proAB region (Pinkham et al., JMB 744, 587-592, 1980). Correlation of a set of such recombinants indicates that the transition may occur from the third T of the core, reading from the left, to about 7 bases farther to the right (Mizuuchi et al., op. cit.). Mizuuchi et al. (op. cit.) have recently carried out an elegant experiment to test directly for the site of crossover in the site-specific integration reaction. In vitro recombination was carried out between unlabeled att POP’ DNA and aft BOB’ DNA carrying one strand labeled with 32P adjacent to one of the 4 bases. After recombination, the labeled BOP’ and POB’ fragments were digested with restriction enzymes, purified and stored to allow accumulation of strand breaks from “P disintegration. Analysis of the fragments generated by the 32P cleavages on an acrylamide sequencing gel provided a series of bands radioactively labeled in the bacterial portion. For instance, for the BOP’ attachment site, the pieces seen as radioactive bands would carry all of P’ (unlabeled) and the portion of labeled B to the right of the 32P disintegration. The P’ piece formed by the disintegration of the 32P closest to the crossover site would carry no B material, and therefore no radioactive label, and would not be seen
Cell 586
at
BOB’
on the gel. By determining the sequence of this missing piece for a series of recombinations using, in turn, each deoxyribonucleotide labeled with 32P, and each strand separately labeled, one could determine the point in the sequence where bacterial information ends and phage att site information begins. The results show that there are two unique crossover points, one on each strand, located as indicated in the figure, resulting in a 5’ overlap. Because the phosphorus atoms rather than the bases were labeled, the results are consistent with either a 5 bp or 7 bp separation between the two crossover points. Only the 7 bp result, however, is consistent with the results obtained from analysis of secondary-site recombination events. Studies on the properties of mutations in the core of att POP’ (called saf, for site-affinity mutations) have helped to define the roles of particular bases in POP’ and BOB’ and the role of homology between the cores in promoting efficient recombination. Weisberg (Mizuuchi et al., op. cit.) obtained such mutants among phages excised from an abnormal attachment site in galT. Phages carrying three changes in the crossover region recombine at one twentieth of normal efficiency with att BOB’; this decrease may reflect changes both in the Int recognition sequences and in homology between the cores. Phages carrying these same three changes recombine tenfold better with the abnormal ga/T att site (from which they were derived, and with which they share 11 of 15 bp of homology in the core) than do wild-type phages (with 8 of 15 bases of the core homologous). If these phage core sequences are important for Int recognition, the positive contribution of homology may be even more important than suggested by this tenfold improvement. It is clear, however, that base homology in the core is not sufficient for site-specific recombination to proceed efficiently; Shulman and Gottesman (op. cit.) recombined BOP’ carrying the core mutation att24 (missing one T in the run of six Ts in the core) with POB’ also carrying att24, and found that the mutant x mutant recombination did not proceed any more efficiently than mutant X wild-type recombination. The core therefore seems to provide both a homologous region for crossovers and essential recognition sequences for the proteins involved in the site-specific recombination reaction.
One can determine the precise location of recognition regions within POP’ and BOB’ sites by determining the protection of these regions from pancreatic DNAase and neocarzinostatin by the proteins involved in the recombination. Int, the first to be purified of the recombination proteins, protects a central core of about 30-35 bases disposed symmetrically around the core in POP’. Only a subset of these bases, including the left-hand portion of the common core, is protected in BOB’. In addition, Int apparently binds to and protects three other sites in POP’: two small sites (15-20 bp) in the P arm, and one larger site (30-35 bp) in the P’ arm (Ross et al., Cell 78, 297-307, 1979; Hsu et al., op. cit.). The P’ arm shows particularly tight binding; only the binding to this site is heparin-resistant. No homology has been seen between the sequences protected in the arms and the core-binding sequence, but some homology can be seen between sites in P and a sequence in the P’protected region. This distinction between core sites and arm sites may suggest that these two different sets of sequences respond to different Int domains (Hsu et al., op. cit.). Since one of the P binding sites and much of the P’ region fall within the limits assigned for POP’ by the DNA “nibbling” experiments described above, these sites may represent the necessary limits of the attachment site. Loss of these protected regions (with retention of the POP’ core region) are sufficient to make POP’ recombine like BOB’; the arm sites therefore must play an essential part in the POP’-BOB’ discrimination by the Int system. The phage X has assigned two gene products, 240 bases of essential site and a variety of regulatory circuits to the job of ensuring efficient integration of its DNA into the host on lysogeny and rapid escape on induction. The crossover event, a staggered cut with a homologous core, leaves both parents intact after a cycle of integration and excision. Current work in a number of laboratories on the interaction of other recombination proteins (host factors and Xis) with the aft sites, as well as studies of mutations in both the core and the arm sites, should fill in the details of this outline. I would like to acknowledge the help of Bob Weisberg. Max Gottesman and Michael Gottesman in commenting uscript.
Howard Nash, on this man-