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Campbell Model
Campbell Model A Campbell, Stanford University, Stanford, CA, USA © 2013 Elsevier Inc. All rights reserved.
Glossary Cointegration Reciprocal recombination between two circular DNA molecules, fusing them into a single molecule. Excision The reversal of integration. General recombination Exchange of DNA between two partners within a segment of extended base sequence identity or similarity (regardless of the nature of that sequence), mediated by enzymes encoded by either the host chromosome or an extrachromosomal element. Insertion sequence (IS) DNA segments that can move from one DNA site to another using enzymes encoded by
Introduction The Campbell model was first proposed to explain the mode of association of the genomes of bacteriophage lambda and its host, Escherichia coli, in lysogenic bacteria. First, the ends of the linear lambda genome become joined, and then the circular phage and host genomes cointegrate by reciprocal crossover within a segment of homology between the two. Such cointe gration (Figure 1) is the essence of the Campbell model, a term that has been applied to all similar cointegrations, whether they take place through site-specific recombination (as in lambda) or by general (homology-dependent) recombination. The reversal of the reaction leads to excision of the integrated element from the chromosome.
Integration by Site-Specific Recombination The validity of the model to lambda integration has been rigorously tested. On entering an infected cell, the lambda genome circularizes by annealing and ligation of complemen tary ‘sticky ends’ (projecting 12-bp single-stranded 5′-ends of the viral DNA). Integration is mediated by a phage-coded protein (integrase) that recognizes specific sequences at the crossover site. One such site is present on each partner (phage and bacterium), although rare integration events use bacterial partner sequences with reduced similarity to the primary sites. Besides integrase, excision requires a second phage-coded pro tein, excisionase. For lambda and E. coli, the segment of sequence identity (123 in Figure 1) is 15 bp long. This is too short to serve as a substrate for general recombinases like E. coli RecA. Among phages and plasmids using lambda-related integrases, the extent of sequence identity at the crossover point varies from as low as 10 to over 100. From mutational and biochemical studies of the integration reaction, the inferred mechanism entails single-strand cleavage at corresponding sites of one DNA strand from each partner, followed by cross-ligation to give a crossed-strand (Holliday) structure. An intermediate in the strand transfer has a covalent
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genes within the movable segment that recognize the end of the segment but not the site of insertion. Integrase An enzyme encoded by an extrachromosomal DNA element that mediates integration of the element into the chromosome. Integration The incorporation of extrachromosomal DNA (such as that of a phage or a plasmid) into the chromosome. Site-specific recombination A reciprocal exchange between two DNA molecules (such as phage and chromosomal DNA) at specific sites on each partner.
DNA–protein link on the 3′-ends of each of the two strands. Subsequently, the other two strands are cleaved and cross-ligated at a position displaced 7 bp from the site of initial cleavage. It is only within the 7 bp between the two sites (over lap segment) that sequence identity between the two partners is required, probably to facilitate strand swapping that takes place between cleavage and ligation. This 7-bp segment is flanked by an approximate reverse repeat apparently used in protein–DNA recognition. The extent of specific sequence needed at the crossover site is about 20 bp. However, in the phage partner, additional specific sequences are needed in the DNA flanking the crossover site (attP). A DNA-binding protein, integration host factor (IHF), is also needed for proper positioning of the DNA loops. A complex (called an intasome) of attP, IHF, and several molecules of integrase forms first. Then bacterial (attB) DNA is recruited. Integrase and excisionase are separately controlled during lambda development so as to give efficient integration in those cells destined for lysogeny and efficient excision within those lysogenic cells that are reinitiating phage production. Phage lambda belongs to a large group of natural (lamb doid) coliphages related to one another in DNA sequence. Lambdoid phages use various integration sites in their host bacteria. Their common feature is an approximate reverse repeat surrounding a 7-bp segment of identity. Some members of the integrase family use a 6 or 8 bp overlap segment, but these integrases are not ordinarily used in phage integration. Some phages and plasmids with little DNA sequence similarity to lambda also use integrases of the same gene family. A phage has also been reported that uses for integration a member of the other major group of site-specific recombinases, the DNA invertase–resolvase family.
Integration by General Recombination When the model was proposed, it was an attempt to provide a unified mechanism for integration by autonomous elements, including most specifically phage lambda and the E. coli ferti lity plasmid F. F integrates mainly by general recombination,
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 1
doi:10.1016/B978-0-12-374984-0.00189-3
Campbell Model
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Abnormal excision of lambda is rare and proceeds by unknown biochemistry that juxtaposes heterologous DNA; with F, the major mechanism is general recombination between homolo gous IS elements flanking the inserted F. Such abnormal elements (called specialized transducing phages and F′ plas mids, respectively) can integrate into the bacterial chromosome by general recombination using the homology provided by the DNA they have picked up from the host. Besides such natural processes, integration by homology has been used extensively with genetically engineered constructs where a host gene is cloned into a phage or plasmid vector. As implied by Figure 1, the resulting integrant has two copies of the cloned segment, in direct orientation, flanking one copy of vector DNA. Where these two copies differ by mutation, excision by general recombination can generate a cell with alleles origin ally present in the cloned insert or vector carrying alleles originally present in the host. Such swaps will occur for alleles at position 2 (Figure 1) if the integrating crossover occurs between 1 and 2 and excision between 2 and 3. 123
yz
Figure 1 Generalized Campbell model. A circular extrachromosomal element (above) integrates into the bacterial chromosome (of which only a linear segment is shown) within homologous DNA (123). abcd and wxyz are genetic markers of element and chromosome, respectively. In phage lambda, the circle is formed by joining the ends of linear DNA (with order abcd) so in the inserted prophage this order is permuted to cdab.
using as portable regions of homology insertion sequences (IS elements) common to both F and the chromosome. Some integration may also take place through replicative transposi tion mediated by the IS elements, a mechanism for cointegrate formation that transcends the Campbell model. Both lambda and F can excise abnormally from the chro mosome to include host DNA adjacent to the insertion site.
See also: Site-specific Recombination.
Further Reading Campbell AM (1962) Episomes. In: Caspari EW and Thoday JM (eds.) Advances in Genetics, vol. II, pp. 101–146. New York: Academic Press. Campbell AM (1992) Chromosomal insertion sites for phages and plasmids. Journal of Bacteriology 174: 7495–7499. Craig NL (1998) The mechanism of conservative site-specific recombination. Annual Review of Genetics 22: 77–106. Nunes-Duby SE, Azaro MA, and Landy A (1995) Swapping DNA strands and sensing homology without branch migration in λ site-specific recombination. Current Biology 5: 139–148. Thorpe HM and Smith MCM (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proceedings of the National Academy of Sciences of the United States of America 95: 5505–5510.
Glossary Cointegration Reciprocal recombination between two circular DNA molecules, fusing them into a single molecule. Excision The reversal of integration. General recombination Exchange of DNA between two partners within a segment of extended base sequence identity or similarity (regardless of the nature of that sequence), mediated by enzymes encoded by either the host chromosome or an extrachromosomal element. Insertion sequence (IS) DNA segments that can move from one DNA site to another using enzymes encoded by
Introduction The Campbell model was first proposed to explain the mode of association of the genomes of bacteriophage lambda and its host, Escherichia coli, in lysogenic bacteria. First, the ends of the linear lambda genome become joined, and then the circular phage and host genomes cointegrate by reciprocal crossover within a segment of homology between the two. Such cointe gration (Figure 1) is the essence of the Campbell model, a term that has been applied to all similar cointegrations, whether they take place through site-specific recombination (as in lambda) or by general (homology-dependent) recombination. The reversal of the reaction leads to excision of the integrated element from the chromosome.
Integration by Site-Specific Recombination The validity of the model to lambda integration has been rigorously tested. On entering an infected cell, the lambda genome circularizes by annealing and ligation of complemen tary ‘sticky ends’ (projecting 12-bp single-stranded 5′-ends of the viral DNA). Integration is mediated by a phage-coded protein (integrase) that recognizes specific sequences at the crossover site. One such site is present on each partner (phage and bacterium), although rare integration events use bacterial partner sequences with reduced similarity to the primary sites. Besides integrase, excision requires a second phage-coded pro tein, excisionase. For lambda and E. coli, the segment of sequence identity (123 in Figure 1) is 15 bp long. This is too short to serve as a substrate for general recombinases like E. coli RecA. Among phages and plasmids using lambda-related integrases, the extent of sequence identity at the crossover point varies from as low as 10 to over 100. From mutational and biochemical studies of the integration reaction, the inferred mechanism entails single-strand cleavage at corresponding sites of one DNA strand from each partner, followed by cross-ligation to give a crossed-strand (Holliday) structure. An intermediate in the strand transfer has a covalent
414
genes within the movable segment that recognize the end of the segment but not the site of insertion. Integrase An enzyme encoded by an extrachromosomal DNA element that mediates integration of the element into the chromosome. Integration The incorporation of extrachromosomal DNA (such as that of a phage or a plasmid) into the chromosome. Site-specific recombination A reciprocal exchange between two DNA molecules (such as phage and chromosomal DNA) at specific sites on each partner.
DNA–protein link on the 3′-ends of each of the two strands. Subsequently, the other two strands are cleaved and cross-ligated at a position displaced 7 bp from the site of initial cleavage. It is only within the 7 bp between the two sites (over lap segment) that sequence identity between the two partners is required, probably to facilitate strand swapping that takes place between cleavage and ligation. This 7-bp segment is flanked by an approximate reverse repeat apparently used in protein–DNA recognition. The extent of specific sequence needed at the crossover site is about 20 bp. However, in the phage partner, additional specific sequences are needed in the DNA flanking the crossover site (attP). A DNA-binding protein, integration host factor (IHF), is also needed for proper positioning of the DNA loops. A complex (called an intasome) of attP, IHF, and several molecules of integrase forms first. Then bacterial (attB) DNA is recruited. Integrase and excisionase are separately controlled during lambda development so as to give efficient integration in those cells destined for lysogeny and efficient excision within those lysogenic cells that are reinitiating phage production. Phage lambda belongs to a large group of natural (lamb doid) coliphages related to one another in DNA sequence. Lambdoid phages use various integration sites in their host bacteria. Their common feature is an approximate reverse repeat surrounding a 7-bp segment of identity. Some members of the integrase family use a 6 or 8 bp overlap segment, but these integrases are not ordinarily used in phage integration. Some phages and plasmids with little DNA sequence similarity to lambda also use integrases of the same gene family. A phage has also been reported that uses for integration a member of the other major group of site-specific recombinases, the DNA invertase–resolvase family.
Integration by General Recombination When the model was proposed, it was an attempt to provide a unified mechanism for integration by autonomous elements, including most specifically phage lambda and the E. coli ferti lity plasmid F. F integrates mainly by general recombination,
Brenner’s Encyclopedia of Genetics, 2nd Edition, Volume 1
doi:10.1016/B978-0-12-374984-0.00189-3
Campbell Model
wx
123
cd
ab
123
cd
wx
123
yz
ab
415
Abnormal excision of lambda is rare and proceeds by unknown biochemistry that juxtaposes heterologous DNA; with F, the major mechanism is general recombination between homolo gous IS elements flanking the inserted F. Such abnormal elements (called specialized transducing phages and F′ plas mids, respectively) can integrate into the bacterial chromosome by general recombination using the homology provided by the DNA they have picked up from the host. Besides such natural processes, integration by homology has been used extensively with genetically engineered constructs where a host gene is cloned into a phage or plasmid vector. As implied by Figure 1, the resulting integrant has two copies of the cloned segment, in direct orientation, flanking one copy of vector DNA. Where these two copies differ by mutation, excision by general recombination can generate a cell with alleles origin ally present in the cloned insert or vector carrying alleles originally present in the host. Such swaps will occur for alleles at position 2 (Figure 1) if the integrating crossover occurs between 1 and 2 and excision between 2 and 3. 123
yz
Figure 1 Generalized Campbell model. A circular extrachromosomal element (above) integrates into the bacterial chromosome (of which only a linear segment is shown) within homologous DNA (123). abcd and wxyz are genetic markers of element and chromosome, respectively. In phage lambda, the circle is formed by joining the ends of linear DNA (with order abcd) so in the inserted prophage this order is permuted to cdab.
using as portable regions of homology insertion sequences (IS elements) common to both F and the chromosome. Some integration may also take place through replicative transposi tion mediated by the IS elements, a mechanism for cointegrate formation that transcends the Campbell model. Both lambda and F can excise abnormally from the chro mosome to include host DNA adjacent to the insertion site.
See also: Site-specific Recombination.
Further Reading Campbell AM (1962) Episomes. In: Caspari EW and Thoday JM (eds.) Advances in Genetics, vol. II, pp. 101–146. New York: Academic Press. Campbell AM (1992) Chromosomal insertion sites for phages and plasmids. Journal of Bacteriology 174: 7495–7499. Craig NL (1998) The mechanism of conservative site-specific recombination. Annual Review of Genetics 22: 77–106. Nunes-Duby SE, Azaro MA, and Landy A (1995) Swapping DNA strands and sensing homology without branch migration in λ site-specific recombination. Current Biology 5: 139–148. Thorpe HM and Smith MCM (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proceedings of the National Academy of Sciences of the United States of America 95: 5505–5510.