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Site-specific inversion: enhancers, recombination proteins, and mechanism
Cell, Vol. 41, 649-650, July 1985, Copyright© 1985 by MIT
0092-8674/851070649-02$02.00/0
Minireview
Site-Specific Inversion: Enhancers, Recombination Proteins, and Mechanism Nancy L. Craig Department of Microbiology and Immunology and the Hooper Research Foundation University of California San Francisco, California 94143
The inversion of DNA segments by site-specific recombination can regulate gene expression (see Plasterk and van de Putte, BBA 782, 111-119, 1984). Inversion of the G segment of phage Mu and the C segment of phages P1 and P7 switches the orientation of tail fiber genes within the invertible segment with respect to an external promoter (panel A), thereby changing the phage host range. Variation of the S. typhimurium flagellar antigens results from inversion of the H segment, which changes the orientation of a promoter within the invertible segment with respect to flagellin genes outside the segment (panel B). Inversion of the P segment of the e14 prophage in the E. coli chromosome can also occur. All of these inversion reactions are closely related: the element-encoded recombinases-Gin, Cin, Hin, and Pin--are highly homologous and can complement each other, and the recombination sites have homologous sequences. In vivo and in vitro analysis of Gin- and Hin-promoted inversion (Kahmann et al., and Johnson and Simon, respectively; Cell, this issue) has revealed the existence of DNA sequences that act as enhancers of site-specific inversion and has shown that several proteins participate in inversion. The Crossover Site The strand exchanges underlying inversion occur within inverted repeats (IRs) flanking an invertible segment (figure). Investigation of the sequences required for Gin- and Hin-promoted inversion suggests that the presence of two IRs is sufficient to promote recombination (although inversion is stimulated by the presence of other sequences-see below). Moreover, the G, C, H, and P IRs share a common sequence. The capacity of the recombinases to substitute for one another suggests that this conserved sequence within the IR may define the minimum crossover site and contain the point of strand exchange. Indeed, Johnson and Simon have shown that the presence of two copies of a 26 bp sequence containing this inversion consensus sequence is sufficient for Hinpromoted inversion. The Enhancer Inversion of minimal substrates containing only the IRs can be greatly stimulated (about 15- to 20-fold) by the presence in cis of additional sequences. These stimulating sequences are within 60 bp segments located about 100-200 bp from one IR (figure). There are probably multiple functional sites within the segments because smaller fragments derived from them have partial activity. The related nature of the Gin and Hin systems certainly suggests that their stimulating sites may be similar, although this has not been directly established. An intriguing feature of these stimulating sequences is that their activity is
independent of their position or orientation within the substrate DNA. For example, the Gin-stimulating segment is active when located almost 3000 bp from either IR. Thus, these recombination stimulating sequences resemble eukaryotic transcriptional enhancers: they act in cis to enhance recombination in a distance- and orientationindependent fashion. Element and Host Recombination Proteins Although the recombinases are necessary for inversion, the presence of purified Gin or Hin protein alone does not promote efficient recombination. The purified recombinase does, however, exhibit a partial activity fundamental to recombination: Johnson and Simon have found that purified Hin protein is a DNA binding protein that can bind specifically to the H segment IRs. Purified Gin and Hin protein require supplementation with a crude extract from cells lacking the recombinase to promote recombination at a high level, indicating that a host component participates in inversion. The stimulatory capacity of the host component is most dramatic when the substrate DNA contains the enhancer: Kahmann et al. find that the host component stimulates Gin-mediated inversion of enhancer-containing substrates about 200-fold, whereas minimal substrates are stimulated about 2-fold. This suggests that the host component acts through the enhancer, although this may not be its exclusive mode of action. Further biochemical and genetic analysis will establish whether the host component and the enhancer have essential or accessory roles in inversion.
--=lnvertible segment =lnveHed repeat 0 =Enhancer p = Promoter G A. ( + )
~
~--[}--
p
,~
Sc Sv
(-)
~.
U U'
Sv
g
gin
m-o--
p
,,
Sc Sv
U'
U
•
Sv
gin
H B. ( O N ) i, p ~
hin
•
i,
H2
rill
H2
rill
(OFF)~ ~ p - -
o.
hin
Cell 650
It has not yet been established whether the host components of the Gin and Hin systems are identical; this would not be surprising given the close relationship of these systems. Kahmann et al. have shown that the host component for Gin-promoted inversion is neither Integration Host Factor, an E. coli protein that plays an accessory role in phage lambda integrase site-specific recombination (Lange-Gustafson and Nash, JBC 259, 12724-12732, 1984), nor protein HU, a prokaryotic histone-like protein. The involvement of a host component in inversion was not anticipated. One reason is that the inversion recombinases are structurally related to resolvase, a site-specific recombinase encoded by Tn3-1ike transposons (see Newman and Grindley, Cell 38, 463-469, 1984), which does not require any other component to promote recombination. The Mechanism of Inversion The emerging picture of site-specific inversion is a complex one: efficient recombination requires two proteins-the element-encoded recombinase and the host component, and two DNA sequences--the two IR sites where strand exchange occurs and the enhancer. How do these elements promote the recognition and juxtaposition of the crossover sites and strand exchange? The finding that Hin protein binds to the crossover site suggests that the inversion recombinases provide specificity determinants, a role that has also been assigned to other site-specific recombinases (Ross et al., Cell 18, 297-307, 1979; Reed and Grindley, Cell 25, 721-728, 1981). These other recombinases also encode the catalytic site for strand exchange: they have sequence-specific topoisomerase activity, which executes the breakage and rejoining reactions that mediate strand exchange. (Reed and Grindiey, op cit; Craig and Nash, Cell 35, 795-803, 1983). Gin- and Hin-promoted inversion occurs efficiently in vitro in the absence of an external high energy source such as ATP, indicating that inversion also occurs through a topoisomerase-like mechanism. It is parsimonious, although premature, to assign both the specificity and strand breakage and rejoining functions in inversion to the recombinase. It is not yet known how the host component and the enhancer promote recombination. A particularly interesting question is how can the enhancer promote recombination in a distance- and orientation-independent fashion? Certainly the same models that have been proposed to account for the action of transcriptional enhancers (see Khoury and Gruss, Cell 33, 313-314, 1983) can be applied to the recombinational enhancers. The enhancer may provide an entry or recognition site for the recombination proteins; it may facilitate inversion by changing the conformation of DNA so that the substrate is more accessible to recombination. The finding that sequences distant from a crossover site can influence recombination is not unprecedented. Recombination sites used by integrase and resolvase require flanking arms up to 150 bp in length (Hsu et al., Nature 285, 85-91, 1980; Kitts et al., EMBO J. 2, 1055-1060, 1983). It should be noted that the effect of altering the positions of these flanking arms has not been examined. It is
known that these arms contain specific binding sites for the recombination proteins but the mechanistic role(s) of these sites has not been defined. One attractive hypothesis is that they are required to form ordered protein-DNA complexes that are the active substrates in recombination (Better et al., PNAS 79, 5837-5841, 1982). The close apposition of binding sites within these arms is consistent with this hypothesis and hints that their positions may not be flexible. Cis-acting enhancers of homologous recombination have also been identified: the Chi sequence of E. coil (see Smith, Cell 34, 709-710, 1983) and the HOT1 sequence of S. cerevisiae (Keil and Roeder, Cell 39, 377-386, 1984). It is tempting to speculate that recombinational enhancers (and perhaps silencers? [Brand et al., Cell 41, 41-48, 1985]) may be involved in other programmed DNA rearrangements such as the assembly of immunoglobulin genes. An important issue in recombination and, indeed, a question that may be fundamentally related to the action of recombinational and transcriptional enhancers,, is how recombination sites are juxtaposed prior to strand exchange. It is clear that in some pathways of recombination, sites are juxtaposed by random collision during three-dimensional diffusion (Mizuuchi et al., JMB 141, 485-494, 1980). However, this is apparently not the only way in which DNA sites can be brought together. A striking feature of the inversion and resolvase reactions is their capacity to execute only intramolecular recombination. Moreover, these recombination systems can ascertain the relative orientation of their recombination sites in a substrate molecule. The inversion systems strongly prefer recombination sites in inverted orientation whereas resolvase strongly prefers sites in direct orientation. It has been suggested that during these reactions the recombination sites are juxtaposed by a tracking mechanism (Abremski et al., Cell 32, 1301-1311, 1983; Kitts et al., op cit.; Krasnow and Cozzarelli, Cell 32, 1313-1324, 1983). This tracking pathway involves the specific binding of the recombination proteins to one recombination site and subsequent scanning of adjacent DNA by this protein-DNA complex. Scanning could occur through one-dimensional diffusion of the complex along the DNA substrate, mediated by nonspecific protein-DNA interactions, until another recombination site is encountered. Outlook Because the inversion enhancers are active in vitro with partially purified recombination proteins, they offer the exciting prospect of examination of their mechanism at the molecular level. Analysis of the host components of inversion will extend our knowledge of the recombination protein repertoire. It is clear that site-specific recombination will continue to provide an attractive arena for dissecting interactions between widely separated DNA sites. Note Added in Proof
A similar enhancement of Cin-promoted inversion can also occur (Huber et al., PNAS 82, 3776-3780, 1985).
0092-8674/851070649-02$02.00/0
Minireview
Site-Specific Inversion: Enhancers, Recombination Proteins, and Mechanism Nancy L. Craig Department of Microbiology and Immunology and the Hooper Research Foundation University of California San Francisco, California 94143
The inversion of DNA segments by site-specific recombination can regulate gene expression (see Plasterk and van de Putte, BBA 782, 111-119, 1984). Inversion of the G segment of phage Mu and the C segment of phages P1 and P7 switches the orientation of tail fiber genes within the invertible segment with respect to an external promoter (panel A), thereby changing the phage host range. Variation of the S. typhimurium flagellar antigens results from inversion of the H segment, which changes the orientation of a promoter within the invertible segment with respect to flagellin genes outside the segment (panel B). Inversion of the P segment of the e14 prophage in the E. coli chromosome can also occur. All of these inversion reactions are closely related: the element-encoded recombinases-Gin, Cin, Hin, and Pin--are highly homologous and can complement each other, and the recombination sites have homologous sequences. In vivo and in vitro analysis of Gin- and Hin-promoted inversion (Kahmann et al., and Johnson and Simon, respectively; Cell, this issue) has revealed the existence of DNA sequences that act as enhancers of site-specific inversion and has shown that several proteins participate in inversion. The Crossover Site The strand exchanges underlying inversion occur within inverted repeats (IRs) flanking an invertible segment (figure). Investigation of the sequences required for Gin- and Hin-promoted inversion suggests that the presence of two IRs is sufficient to promote recombination (although inversion is stimulated by the presence of other sequences-see below). Moreover, the G, C, H, and P IRs share a common sequence. The capacity of the recombinases to substitute for one another suggests that this conserved sequence within the IR may define the minimum crossover site and contain the point of strand exchange. Indeed, Johnson and Simon have shown that the presence of two copies of a 26 bp sequence containing this inversion consensus sequence is sufficient for Hinpromoted inversion. The Enhancer Inversion of minimal substrates containing only the IRs can be greatly stimulated (about 15- to 20-fold) by the presence in cis of additional sequences. These stimulating sequences are within 60 bp segments located about 100-200 bp from one IR (figure). There are probably multiple functional sites within the segments because smaller fragments derived from them have partial activity. The related nature of the Gin and Hin systems certainly suggests that their stimulating sites may be similar, although this has not been directly established. An intriguing feature of these stimulating sequences is that their activity is
independent of their position or orientation within the substrate DNA. For example, the Gin-stimulating segment is active when located almost 3000 bp from either IR. Thus, these recombination stimulating sequences resemble eukaryotic transcriptional enhancers: they act in cis to enhance recombination in a distance- and orientationindependent fashion. Element and Host Recombination Proteins Although the recombinases are necessary for inversion, the presence of purified Gin or Hin protein alone does not promote efficient recombination. The purified recombinase does, however, exhibit a partial activity fundamental to recombination: Johnson and Simon have found that purified Hin protein is a DNA binding protein that can bind specifically to the H segment IRs. Purified Gin and Hin protein require supplementation with a crude extract from cells lacking the recombinase to promote recombination at a high level, indicating that a host component participates in inversion. The stimulatory capacity of the host component is most dramatic when the substrate DNA contains the enhancer: Kahmann et al. find that the host component stimulates Gin-mediated inversion of enhancer-containing substrates about 200-fold, whereas minimal substrates are stimulated about 2-fold. This suggests that the host component acts through the enhancer, although this may not be its exclusive mode of action. Further biochemical and genetic analysis will establish whether the host component and the enhancer have essential or accessory roles in inversion.
--=lnvertible segment =lnveHed repeat 0 =Enhancer p = Promoter G A. ( + )
~
~--[}--
p
,~
Sc Sv
(-)
~.
U U'
Sv
g
gin
m-o--
p
,,
Sc Sv
U'
U
•
Sv
gin
H B. ( O N ) i, p ~
hin
•
i,
H2
rill
H2
rill
(OFF)~ ~ p - -
o.
hin
Cell 650
It has not yet been established whether the host components of the Gin and Hin systems are identical; this would not be surprising given the close relationship of these systems. Kahmann et al. have shown that the host component for Gin-promoted inversion is neither Integration Host Factor, an E. coli protein that plays an accessory role in phage lambda integrase site-specific recombination (Lange-Gustafson and Nash, JBC 259, 12724-12732, 1984), nor protein HU, a prokaryotic histone-like protein. The involvement of a host component in inversion was not anticipated. One reason is that the inversion recombinases are structurally related to resolvase, a site-specific recombinase encoded by Tn3-1ike transposons (see Newman and Grindley, Cell 38, 463-469, 1984), which does not require any other component to promote recombination. The Mechanism of Inversion The emerging picture of site-specific inversion is a complex one: efficient recombination requires two proteins-the element-encoded recombinase and the host component, and two DNA sequences--the two IR sites where strand exchange occurs and the enhancer. How do these elements promote the recognition and juxtaposition of the crossover sites and strand exchange? The finding that Hin protein binds to the crossover site suggests that the inversion recombinases provide specificity determinants, a role that has also been assigned to other site-specific recombinases (Ross et al., Cell 18, 297-307, 1979; Reed and Grindley, Cell 25, 721-728, 1981). These other recombinases also encode the catalytic site for strand exchange: they have sequence-specific topoisomerase activity, which executes the breakage and rejoining reactions that mediate strand exchange. (Reed and Grindiey, op cit; Craig and Nash, Cell 35, 795-803, 1983). Gin- and Hin-promoted inversion occurs efficiently in vitro in the absence of an external high energy source such as ATP, indicating that inversion also occurs through a topoisomerase-like mechanism. It is parsimonious, although premature, to assign both the specificity and strand breakage and rejoining functions in inversion to the recombinase. It is not yet known how the host component and the enhancer promote recombination. A particularly interesting question is how can the enhancer promote recombination in a distance- and orientation-independent fashion? Certainly the same models that have been proposed to account for the action of transcriptional enhancers (see Khoury and Gruss, Cell 33, 313-314, 1983) can be applied to the recombinational enhancers. The enhancer may provide an entry or recognition site for the recombination proteins; it may facilitate inversion by changing the conformation of DNA so that the substrate is more accessible to recombination. The finding that sequences distant from a crossover site can influence recombination is not unprecedented. Recombination sites used by integrase and resolvase require flanking arms up to 150 bp in length (Hsu et al., Nature 285, 85-91, 1980; Kitts et al., EMBO J. 2, 1055-1060, 1983). It should be noted that the effect of altering the positions of these flanking arms has not been examined. It is
known that these arms contain specific binding sites for the recombination proteins but the mechanistic role(s) of these sites has not been defined. One attractive hypothesis is that they are required to form ordered protein-DNA complexes that are the active substrates in recombination (Better et al., PNAS 79, 5837-5841, 1982). The close apposition of binding sites within these arms is consistent with this hypothesis and hints that their positions may not be flexible. Cis-acting enhancers of homologous recombination have also been identified: the Chi sequence of E. coil (see Smith, Cell 34, 709-710, 1983) and the HOT1 sequence of S. cerevisiae (Keil and Roeder, Cell 39, 377-386, 1984). It is tempting to speculate that recombinational enhancers (and perhaps silencers? [Brand et al., Cell 41, 41-48, 1985]) may be involved in other programmed DNA rearrangements such as the assembly of immunoglobulin genes. An important issue in recombination and, indeed, a question that may be fundamentally related to the action of recombinational and transcriptional enhancers,, is how recombination sites are juxtaposed prior to strand exchange. It is clear that in some pathways of recombination, sites are juxtaposed by random collision during three-dimensional diffusion (Mizuuchi et al., JMB 141, 485-494, 1980). However, this is apparently not the only way in which DNA sites can be brought together. A striking feature of the inversion and resolvase reactions is their capacity to execute only intramolecular recombination. Moreover, these recombination systems can ascertain the relative orientation of their recombination sites in a substrate molecule. The inversion systems strongly prefer recombination sites in inverted orientation whereas resolvase strongly prefers sites in direct orientation. It has been suggested that during these reactions the recombination sites are juxtaposed by a tracking mechanism (Abremski et al., Cell 32, 1301-1311, 1983; Kitts et al., op cit.; Krasnow and Cozzarelli, Cell 32, 1313-1324, 1983). This tracking pathway involves the specific binding of the recombination proteins to one recombination site and subsequent scanning of adjacent DNA by this protein-DNA complex. Scanning could occur through one-dimensional diffusion of the complex along the DNA substrate, mediated by nonspecific protein-DNA interactions, until another recombination site is encountered. Outlook Because the inversion enhancers are active in vitro with partially purified recombination proteins, they offer the exciting prospect of examination of their mechanism at the molecular level. Analysis of the host components of inversion will extend our knowledge of the recombination protein repertoire. It is clear that site-specific recombination will continue to provide an attractive arena for dissecting interactions between widely separated DNA sites. Note Added in Proof
A similar enhancement of Cin-promoted inversion can also occur (Huber et al., PNAS 82, 3776-3780, 1985).