Cell, Vol. 68, 1101-I 108, March 20. 1992. Copyright
0 1992 by Cell Press
The Mu Transpositional Enhancer Can Function in Trans: Requirement of the Enhancer for Synapsis But Not Strand Cleavage Michael G. Surette’ and George Chaconas Department of Biochemistry University of Western Ontario LoIndon, Ontario N6A 5Cl Canada
Summary The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer. Introduction Bacteriophage Mu has provided a productive system for the study of DNA transposition (see Pato, 1989, for a recent review). Much of our understanding of the details of Mu transposition at the molecular level has been the offspring of a soluble in vitro transposition system developed by Mizuuchi (1983). Although Mu is perhaps the best understood mobile genetic element, much still remains to be uncovered about the molecular details of the Mu DNA transposition process. The early steps in the transposition of a mini-Mu are characterized by the formation of stable protein-DNA complexes (Surette et al., 1987; Craigie and Mizuuchi, 1987). These reactions are summarized in Figure 1. In a Type 1 complex, also known as a cleaved donor complex, the left and right Mu ends are brought together and held in a noncovalent protein-DNA complex (Surette et al., 1987; Craigie and Mizuuchi, 1987). Formation of this complex is dependent on the introduction of nicks at the 3’ ends of the Mu DNA (Surette et al., 1991). Addition of MuB, ATP, and non-Mu target DNA results in the strand transfer of the free 3’ Mu ends to the target DNA and the generation of a second stable noncovalent protein-DNA complex, a Type 2 complex or strand transfer complex (Surette et al., 1987; Craigie and Mizuuchi, 1987). The protein-free form of this product is a 8 structure (Craigie and Mizuuchi, 1985; Miller and Chaconas, 1986), the pre*Present address: Department of Molecular Laboratory, Princeton, New Jersey, 08544
Biology, Lewis Thomas
dieted product of transposition via the Shapiro model of transposition (Shapiro, 1979; Arthur and Sherratt, 1979). After the initial characterization of these Mu transposition complexes or transposomes, it was determined that in addition to the Mu ends, a third Mu DNA site was required for the formation of the Type 1 complex (Mizuuchi and Mizuuchi, 1989; Leung et al., 1989; Surette et al., 1989). This enhancer-like element, also referred to as the internal activating site, was found between the two Mu ends and, in its natural position, is located about 950 bp from the Mu left end. The functional enhancer site is composed of two operators from the Mu early region (01 and 02) that flank an integration host factor (IHF) binding site. Binding of MuA protein to 01 and 02 is required for enhancer function, and separate domains of the MuA protein bind to the Mu ends and the enhancer (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989). IHF binding to the enhancer reduces the concentration of MuA and HU proteins required for the in vitro reaction and alao allows the reaction to proceed at reduced levels of mini-Mu donor supercoiling (Surette et al., 1989; Surette, 1990). The transpositional enhancer was shown to function in a distanceindependent manner (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989; Surette et al., 1989) and to a limited extent in the reverse orientation in the presence of IHF (Surette et al., 1989). The enhancer element does not function if placed outside of the Mu ends on a mini-Mu donor plasmid (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989). The role of the enhancer in Mu DNA transposition has been demonstrated to becloselycoupled with the topological restrictions governing the Mu DNA strand transfer reaction. Mizuuchi and Mizuuchi (1989) have demonstrated that under reaction conditions that alleviate the topological specificity of the reaction (Craigie and Mizuuchi, 1986) there is no longer a requirement for the enhancer element; the reaction no longer requires a supercoiled substrate or Mu ends in their properorientation. Moreover, mutant MuA that can bind the Mu ends but is unable to bind the enhancer site is capable of promoting the reaction under these conditions (Mizuuchi and Mizuuchi, 1989). Sitespecific recombination in DNA inversion systems (see Glasgow et al., 1989, for review) is similar in that an enhancer element is required in addition to the two recombination sites. lnvertase mutants that are enhancer independent have been isolated for the Gin and Cin systems (Klippel et al., 1988; Haffter and Bickle, 1988); these mutant recombinases also have relaxed topological specificity. In addition, they are still capable of interacting with the enhancer, and when it is present, the third DNA site can still impose topological restraints on the reaction (Klippel et al., 1988). These results strongly implicate the enhancer in the mechanism of sensing the relative orientation of the two recombination sites, and are consistent with models in which three site synapsis is an essential early event in the reaction (Johnson et al., 1987; Klippel et al., 1988; Haffter and Bickle, 1988; Kanaar et al., 1989). Three site
Cell 1102
Mini-Mu donor plasmid
TYPO 2
TYPO 1
Transpososome
Transpososome
Enhancer SDS
IlliF) A,
9, ATP, M92+
HU, Mg2+>
Strand
Cleavage
transfer’
SDS
SDS
I
V -
Non-Mu donor seq”ences
=
Mu sequences
~
Target sequences
-
Nicked
Figure 1. The Mu DNA Strand Transfer
8 Structure u
donor
(RI-
target
(R)-
donor
ISI-
target
(Sl-
TYPE 2
e >
Reaction
A Type 1 complex (also known as a cleaved donor complex), which is an intermediate in the strand transfer reaction, is formed when a supercoiled mini-Mu donor plasmid is incubated with the MuA protein and Eschericia coli HU protein (Surette et al., 1987; Craigie and Mizuuchi, 1987). The Mu ends are held together in a higher order proteinDNA complex (Type 1 transpososome) defining two topological domains: a relaxed non-Mu domain and a supercoiled Mu domain. Disruption of the complex with SDS results in the liberation of a nicked donor plasmid. The Type 2 complex (also called the strand transfer complex) is the product of the strand transfer reaction that remains complexed with protein (Type 2 transpososome). In addition to Type 1 reaction requirements, MuB protein, ATP, and target DNA are required for Type 2 complex formation. Disruption of the Type 2 complex with SDS liberates the protein-free strand-transferred product or 8 structure (Miller and Chaconas, 1988; Craigie and Mizuuchi, 1985). Although the 9 structure above has been drawn as a relaxed molecule for simplicity of presentation, it is important to note that the Mu DNA sequences, but not the vector or target DNA, are topologically constrained (Craigie and Mizuuchi, 1985). Hence the Mu DNA stem retains the Mu supercoils originally present in the Type 2 complex. This figure was adapted from Surette et al. (1987).
synaptic complexes have been directly observed in the Hin system (Heichman and Johnson, 1990). In this work, we have further characterized the role of the enhancer-like element in the in vitro strand transfer reaction of a mini-Mu donor plasmid. We demonstrate that the enhancer function can be provided in trans on an unlinked linear DNA molecule. Furthermore, we demonstrate that the role of the enhancer is confined to promoting the correct synapsis of the left and right Mu ends and is not required for any of the subsequent steps, i.e., strand cleavage at the Mu ends and strand transfer to target DNA. Results A Linear Mu Enhancer Element Can Function in Trans To investigate the role of the transpositional enhancer in theearlystepsof mini-Mu transposition in vitro, we isolated a 199 bp DNA fragment that contained all the DNA in the enhancer region that was required in cis for enhancer function. This fragment was assayed for the ability to provide enhancer function in trans. The results are presented in Figure 2. The mini-Mu plasmid pGG215, which has the enhancer element located in cis at its usual position,
Figure 2. The Mu Transpositional EnhancerCan in the In Vitro Strand Transfer Reaction
Be Provided in Trans
Ethidium bromide-stained 1% agarose gel showing the products of in vitro strand transfer reactions carried out as described in Experimental Procedures. Samples wereelectrophoresed in theabsence (-)orpresence (+) of SDS. The position of the Type 2 complex and the proteinfree 9 structure are indicated on the right. The mini-Mu donor pGG215 contains both Mu ends and the enhancer-like element. Plasmid pMS2B2 has the enhancer site deleted. Note that the 9 topoisomers generated with pMS2B2 did not comigrate exactly with those derived from pGG215. owing to the slight difference in size between the two mini-Mu plasmids. The 199 bp enhancer fragment when present was at a 40 molar excess over the mini-Mu plasmid. This fragment migrated off the bottom of the gel under these conditions. All reactions contained IHF and were incubated at 30% for 45 min.
formed Type 2 complexes very efficiently. As shown previously (Surette et al., 1989) deletion of the enhancer region from this plasmid to generate pMS2B2 resulted in a mini-Mu that did not function in the in vitro strand transfer reaction under our normal reaction conditions. However, when an enhancer fragment was added in trans to a reaction containing pMS2B2, Type 2 complex was detected (about 50% reaction in 45 min). Biochemical analysis of the product of this reaction demonstrated that it was the predicted 8 structure for a normal strand transfer reaction in which both Mu ends had been transferred to target DNA (data not shown). The protein-free 0 structures (+SDS lanes) migrated as distinct topoisomers. The reaction with the enhancer in trans was less efficient than with the enhancer in cis; however, in the presence of an excess of the enhancer DNA and with incubation times of 90 min, the reaction could be driven to greater than 90% conversion of the input substrate. Increasing the concentration of A protein in the reaction did not stimulate the reaction with the enhancer in trans, but it was important to maintain a 1.5 x molar ratio of IHF to input enhancer. The results demonstrate that a linear enhancer can function in trans
Mu Enhancer 1103
Function
In Trans
Table 1. Topological Specificity Enhancer In Cis and In Trans -
of Strand Transfer
Reaction with
In Vitro Reactionb
Mini-Mu Donor
Description
Topological State’
pGG215
wild type
S R L
cis
~MB216~
right end inversion
S R L
cis
-
pMS2B2”
A enhancer
S R L
trans
-
Figure 3. The Formation of Type 2 Complexes Function of Enhancer Concentration
-
The reaction of pMS2B2 was measured in reactions containing MuA, HU,andIHFinthepresenceofvariousamountsofthe199bpenhancer fragment. Reactions were carried at 30°C for 45 min, and the extent of reaction was determined by densitometric analysis of an ethidium bromide-stained gel as described in Experimental Procedures.
pMS2821’
A enhancer + right end
S R L
Enhancer
-IHF
+IHF
>95%”
>95%”
-5o%g
trans
-
a S, R, and L refer to supercoiled, relaxed, and linear forms of the plasmids. b Both Type 1 and Type 2 reactions gave similar results. Negative (-) symbol indicates < 0.3% reaction. c The reaction was essentially complete by 20 min at 30%. d pMB216 is a derivative of the wild-type mini-Mu pGG215 in which the orientation of the Pstl fragment carrying the Mu right end has been reversed (Lavoie and Chaconas, 1990). e pMS2B2 is a pGG215 derivative with a 792 bp deletion (Nsi-Ball) removing the entire enhancer region (Surette et al., 1989). ’ pMS2B21 carries both the enhancer deletion and right end inversion (see Experimental Procedures). 9 The extent of reaction with a 50 molar excess of enhancer for 45-60 min at 30%.
on an unlinked DNA molecule when the left and right Mu ends are both present on a supercoiled plasmid. Transposition of mini-Mu donor plasmids normally requires that the Mu ends be in their natural orientation both in vivo (Schumm and Howe, 1981) and in vitro (Mizuuchi, 1983) and on a supercoiled plasmid in the in vitro reaction (Mizuuchi, 1983). The topological requirements and the requirement for IHF for the reaction of a mini-Mu plasmid with the enhancer element in cis or in trans are summarized in Table 1. The orientation specificity of the Mu ends and the requirement for supercoiling of the mini-Mu donor plasmid were not relieved by placing the enhancer in trans; no reaction was detected if the ends were not in their proper orientation, and no reaction products were detected if the mini-Mu was relaxed or linear. Furthermore, the reaction with the enhancer in trans was dependent on the presence of IHF. Requirements for Enhancer Function in Trans The reactions presented above for the enhancer in trans utilized a small fragment that was present at a 50-fold molar excess. As shown in Figure 3, the maximal stimulation of the reaction of pMS2B2 by this enhancer fragment was saturating under these conditions. Higher concentrations of MuA did not increase the extent of the reaction, nor did higher concentrations of the enhancer fragment. However, even at a molar ratio of enhancer to mini-Mu was readily donor of two, a low level of reaction (3%~5%)
with pMS2B2
as a
detected after a45 min incubation. Under saturating conditions, the initial rate of reaction with the enhancer in trans was 20- to 25-fold slower than with the enhancer in cis (data not shown). We assayed a variety of DNA molecules with an enhancer element for their ability to provide enhancer function in trans (Figure 4). The reactions were carried out at 20 and 50 molar excess of enhancer relative to the miniMu for 45 min. The results are expressed as percent of pMS2B2 DNA converted to Type 2 complex. Under these
“”
I
A
Figure 4. Efficiency
B
C
D
E
of Various Enhancer
F
G
Fragments
H
I
in Trans
The top panel shows the various enhancer-containing DNAs assayed for their ability to promote Type 2 complex formation with pMS2B2. The length of DNA flanking each end of the enhancer (except those carried on closed circular plasmids) is indicated to the left and right of the drawings, The size of pMS357 is 2.86 kilobase pairs. On the lower panel, the percent reaction determined as described in the Experimental Procedures at 20 and 50 molar excess of the enhancer fragment is shown.
Cell 1104
same reaction conditions, pGG215 (i.e., enhancer in cis) was converted to greater than 9 5 % Type 2 in 20 min. As demonstrated above, no reaction was detected (
0 1992 by Cell Press
The Mu Transpositional Enhancer Can Function in Trans: Requirement of the Enhancer for Synapsis But Not Strand Cleavage Michael G. Surette’ and George Chaconas Department of Biochemistry University of Western Ontario LoIndon, Ontario N6A 5Cl Canada
Summary The phage Mu transpositional enhancer has been previously shown to stimulate the initial rate of the Mu DNA strand transfer reaction by a factor of 100. We now show that the Mu enhancer can function in trans on an unlinked DNA molecule. This activity is greatly facilitated by the presence of a free DNA end proximal to the enhancer element. Function of the enhancer in trans does not alter either the requirement for donor DNA supercoiling or for the two Mu ends to be in their proper orientation on the donor plasmid. An important consequence of these findings is that we have been able to evaluate directly the step in the transposition reaction for which the enhancer is required. We show that the role of the enhancer is limited to promoting productive synapsis; efficient strand cleavage can occur in the absence of the enhancer. Introduction Bacteriophage Mu has provided a productive system for the study of DNA transposition (see Pato, 1989, for a recent review). Much of our understanding of the details of Mu transposition at the molecular level has been the offspring of a soluble in vitro transposition system developed by Mizuuchi (1983). Although Mu is perhaps the best understood mobile genetic element, much still remains to be uncovered about the molecular details of the Mu DNA transposition process. The early steps in the transposition of a mini-Mu are characterized by the formation of stable protein-DNA complexes (Surette et al., 1987; Craigie and Mizuuchi, 1987). These reactions are summarized in Figure 1. In a Type 1 complex, also known as a cleaved donor complex, the left and right Mu ends are brought together and held in a noncovalent protein-DNA complex (Surette et al., 1987; Craigie and Mizuuchi, 1987). Formation of this complex is dependent on the introduction of nicks at the 3’ ends of the Mu DNA (Surette et al., 1991). Addition of MuB, ATP, and non-Mu target DNA results in the strand transfer of the free 3’ Mu ends to the target DNA and the generation of a second stable noncovalent protein-DNA complex, a Type 2 complex or strand transfer complex (Surette et al., 1987; Craigie and Mizuuchi, 1987). The protein-free form of this product is a 8 structure (Craigie and Mizuuchi, 1985; Miller and Chaconas, 1986), the pre*Present address: Department of Molecular Laboratory, Princeton, New Jersey, 08544
Biology, Lewis Thomas
dieted product of transposition via the Shapiro model of transposition (Shapiro, 1979; Arthur and Sherratt, 1979). After the initial characterization of these Mu transposition complexes or transposomes, it was determined that in addition to the Mu ends, a third Mu DNA site was required for the formation of the Type 1 complex (Mizuuchi and Mizuuchi, 1989; Leung et al., 1989; Surette et al., 1989). This enhancer-like element, also referred to as the internal activating site, was found between the two Mu ends and, in its natural position, is located about 950 bp from the Mu left end. The functional enhancer site is composed of two operators from the Mu early region (01 and 02) that flank an integration host factor (IHF) binding site. Binding of MuA protein to 01 and 02 is required for enhancer function, and separate domains of the MuA protein bind to the Mu ends and the enhancer (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989). IHF binding to the enhancer reduces the concentration of MuA and HU proteins required for the in vitro reaction and alao allows the reaction to proceed at reduced levels of mini-Mu donor supercoiling (Surette et al., 1989; Surette, 1990). The transpositional enhancer was shown to function in a distanceindependent manner (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989; Surette et al., 1989) and to a limited extent in the reverse orientation in the presence of IHF (Surette et al., 1989). The enhancer element does not function if placed outside of the Mu ends on a mini-Mu donor plasmid (Leung et al., 1989; Mizuuchi and Mizuuchi, 1989). The role of the enhancer in Mu DNA transposition has been demonstrated to becloselycoupled with the topological restrictions governing the Mu DNA strand transfer reaction. Mizuuchi and Mizuuchi (1989) have demonstrated that under reaction conditions that alleviate the topological specificity of the reaction (Craigie and Mizuuchi, 1986) there is no longer a requirement for the enhancer element; the reaction no longer requires a supercoiled substrate or Mu ends in their properorientation. Moreover, mutant MuA that can bind the Mu ends but is unable to bind the enhancer site is capable of promoting the reaction under these conditions (Mizuuchi and Mizuuchi, 1989). Sitespecific recombination in DNA inversion systems (see Glasgow et al., 1989, for review) is similar in that an enhancer element is required in addition to the two recombination sites. lnvertase mutants that are enhancer independent have been isolated for the Gin and Cin systems (Klippel et al., 1988; Haffter and Bickle, 1988); these mutant recombinases also have relaxed topological specificity. In addition, they are still capable of interacting with the enhancer, and when it is present, the third DNA site can still impose topological restraints on the reaction (Klippel et al., 1988). These results strongly implicate the enhancer in the mechanism of sensing the relative orientation of the two recombination sites, and are consistent with models in which three site synapsis is an essential early event in the reaction (Johnson et al., 1987; Klippel et al., 1988; Haffter and Bickle, 1988; Kanaar et al., 1989). Three site
Cell 1102
Mini-Mu donor plasmid
TYPO 2
TYPO 1
Transpososome
Transpososome
Enhancer SDS
IlliF) A,
9, ATP, M92+
HU, Mg2+>
Strand
Cleavage
transfer’
SDS
SDS
I
V -
Non-Mu donor seq”ences
=
Mu sequences
~
Target sequences
-
Nicked
Figure 1. The Mu DNA Strand Transfer
8 Structure u
donor
(RI-
target
(R)-
donor
ISI-
target
(Sl-
TYPE 2
e >
Reaction
A Type 1 complex (also known as a cleaved donor complex), which is an intermediate in the strand transfer reaction, is formed when a supercoiled mini-Mu donor plasmid is incubated with the MuA protein and Eschericia coli HU protein (Surette et al., 1987; Craigie and Mizuuchi, 1987). The Mu ends are held together in a higher order proteinDNA complex (Type 1 transpososome) defining two topological domains: a relaxed non-Mu domain and a supercoiled Mu domain. Disruption of the complex with SDS results in the liberation of a nicked donor plasmid. The Type 2 complex (also called the strand transfer complex) is the product of the strand transfer reaction that remains complexed with protein (Type 2 transpososome). In addition to Type 1 reaction requirements, MuB protein, ATP, and target DNA are required for Type 2 complex formation. Disruption of the Type 2 complex with SDS liberates the protein-free strand-transferred product or 8 structure (Miller and Chaconas, 1988; Craigie and Mizuuchi, 1985). Although the 9 structure above has been drawn as a relaxed molecule for simplicity of presentation, it is important to note that the Mu DNA sequences, but not the vector or target DNA, are topologically constrained (Craigie and Mizuuchi, 1985). Hence the Mu DNA stem retains the Mu supercoils originally present in the Type 2 complex. This figure was adapted from Surette et al. (1987).
synaptic complexes have been directly observed in the Hin system (Heichman and Johnson, 1990). In this work, we have further characterized the role of the enhancer-like element in the in vitro strand transfer reaction of a mini-Mu donor plasmid. We demonstrate that the enhancer function can be provided in trans on an unlinked linear DNA molecule. Furthermore, we demonstrate that the role of the enhancer is confined to promoting the correct synapsis of the left and right Mu ends and is not required for any of the subsequent steps, i.e., strand cleavage at the Mu ends and strand transfer to target DNA. Results A Linear Mu Enhancer Element Can Function in Trans To investigate the role of the transpositional enhancer in theearlystepsof mini-Mu transposition in vitro, we isolated a 199 bp DNA fragment that contained all the DNA in the enhancer region that was required in cis for enhancer function. This fragment was assayed for the ability to provide enhancer function in trans. The results are presented in Figure 2. The mini-Mu plasmid pGG215, which has the enhancer element located in cis at its usual position,
Figure 2. The Mu Transpositional EnhancerCan in the In Vitro Strand Transfer Reaction
Be Provided in Trans
Ethidium bromide-stained 1% agarose gel showing the products of in vitro strand transfer reactions carried out as described in Experimental Procedures. Samples wereelectrophoresed in theabsence (-)orpresence (+) of SDS. The position of the Type 2 complex and the proteinfree 9 structure are indicated on the right. The mini-Mu donor pGG215 contains both Mu ends and the enhancer-like element. Plasmid pMS2B2 has the enhancer site deleted. Note that the 9 topoisomers generated with pMS2B2 did not comigrate exactly with those derived from pGG215. owing to the slight difference in size between the two mini-Mu plasmids. The 199 bp enhancer fragment when present was at a 40 molar excess over the mini-Mu plasmid. This fragment migrated off the bottom of the gel under these conditions. All reactions contained IHF and were incubated at 30% for 45 min.
formed Type 2 complexes very efficiently. As shown previously (Surette et al., 1989) deletion of the enhancer region from this plasmid to generate pMS2B2 resulted in a mini-Mu that did not function in the in vitro strand transfer reaction under our normal reaction conditions. However, when an enhancer fragment was added in trans to a reaction containing pMS2B2, Type 2 complex was detected (about 50% reaction in 45 min). Biochemical analysis of the product of this reaction demonstrated that it was the predicted 8 structure for a normal strand transfer reaction in which both Mu ends had been transferred to target DNA (data not shown). The protein-free 0 structures (+SDS lanes) migrated as distinct topoisomers. The reaction with the enhancer in trans was less efficient than with the enhancer in cis; however, in the presence of an excess of the enhancer DNA and with incubation times of 90 min, the reaction could be driven to greater than 90% conversion of the input substrate. Increasing the concentration of A protein in the reaction did not stimulate the reaction with the enhancer in trans, but it was important to maintain a 1.5 x molar ratio of IHF to input enhancer. The results demonstrate that a linear enhancer can function in trans
Mu Enhancer 1103
Function
In Trans
Table 1. Topological Specificity Enhancer In Cis and In Trans -
of Strand Transfer
Reaction with
In Vitro Reactionb
Mini-Mu Donor
Description
Topological State’
pGG215
wild type
S R L
cis
~MB216~
right end inversion
S R L
cis
-
pMS2B2”
A enhancer
S R L
trans
-
Figure 3. The Formation of Type 2 Complexes Function of Enhancer Concentration
-
The reaction of pMS2B2 was measured in reactions containing MuA, HU,andIHFinthepresenceofvariousamountsofthe199bpenhancer fragment. Reactions were carried at 30°C for 45 min, and the extent of reaction was determined by densitometric analysis of an ethidium bromide-stained gel as described in Experimental Procedures.
pMS2821’
A enhancer + right end
S R L
Enhancer
-IHF
+IHF
>95%”
>95%”
-5o%g
trans
-
a S, R, and L refer to supercoiled, relaxed, and linear forms of the plasmids. b Both Type 1 and Type 2 reactions gave similar results. Negative (-) symbol indicates < 0.3% reaction. c The reaction was essentially complete by 20 min at 30%. d pMB216 is a derivative of the wild-type mini-Mu pGG215 in which the orientation of the Pstl fragment carrying the Mu right end has been reversed (Lavoie and Chaconas, 1990). e pMS2B2 is a pGG215 derivative with a 792 bp deletion (Nsi-Ball) removing the entire enhancer region (Surette et al., 1989). ’ pMS2B21 carries both the enhancer deletion and right end inversion (see Experimental Procedures). 9 The extent of reaction with a 50 molar excess of enhancer for 45-60 min at 30%.
on an unlinked DNA molecule when the left and right Mu ends are both present on a supercoiled plasmid. Transposition of mini-Mu donor plasmids normally requires that the Mu ends be in their natural orientation both in vivo (Schumm and Howe, 1981) and in vitro (Mizuuchi, 1983) and on a supercoiled plasmid in the in vitro reaction (Mizuuchi, 1983). The topological requirements and the requirement for IHF for the reaction of a mini-Mu plasmid with the enhancer element in cis or in trans are summarized in Table 1. The orientation specificity of the Mu ends and the requirement for supercoiling of the mini-Mu donor plasmid were not relieved by placing the enhancer in trans; no reaction was detected if the ends were not in their proper orientation, and no reaction products were detected if the mini-Mu was relaxed or linear. Furthermore, the reaction with the enhancer in trans was dependent on the presence of IHF. Requirements for Enhancer Function in Trans The reactions presented above for the enhancer in trans utilized a small fragment that was present at a 50-fold molar excess. As shown in Figure 3, the maximal stimulation of the reaction of pMS2B2 by this enhancer fragment was saturating under these conditions. Higher concentrations of MuA did not increase the extent of the reaction, nor did higher concentrations of the enhancer fragment. However, even at a molar ratio of enhancer to mini-Mu was readily donor of two, a low level of reaction (3%~5%)
with pMS2B2
as a
detected after a45 min incubation. Under saturating conditions, the initial rate of reaction with the enhancer in trans was 20- to 25-fold slower than with the enhancer in cis (data not shown). We assayed a variety of DNA molecules with an enhancer element for their ability to provide enhancer function in trans (Figure 4). The reactions were carried out at 20 and 50 molar excess of enhancer relative to the miniMu for 45 min. The results are expressed as percent of pMS2B2 DNA converted to Type 2 complex. Under these
“”
I
A
Figure 4. Efficiency
B
C
D
E
of Various Enhancer
F
G
Fragments
H
I
in Trans
The top panel shows the various enhancer-containing DNAs assayed for their ability to promote Type 2 complex formation with pMS2B2. The length of DNA flanking each end of the enhancer (except those carried on closed circular plasmids) is indicated to the left and right of the drawings, The size of pMS357 is 2.86 kilobase pairs. On the lower panel, the percent reaction determined as described in the Experimental Procedures at 20 and 50 molar excess of the enhancer fragment is shown.
Cell 1104
same reaction conditions, pGG215 (i.e., enhancer in cis) was converted to greater than 9 5 % Type 2 in 20 min. As demonstrated above, no reaction was detected (
