Nov 22, 1992 - Anke Klippel1'2, Roland Kanaar,. Regine Kahmann',3 and Nicholas R.Cozzarelli ... DNA supercoiling (Bliska and Cozzarelli, 1987; Boles et al.,.
The EMBO Journal vol. 12 no.3 pp. 1047 - 1057, 1993
Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants
Anke Klippel1'2, Roland Kanaar, Regine Kahmann',3 and Nicholas R.Cozzarelli Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, 401 Barker Hall, University of California, Berkeley CA 94720, USA and lInstitut fur Genbiologische Forschung Berlin GmbH, D-1000 Berlin 33, Germany 2Present address: Howard Hughes Medical Institute and Cardiovascular Research Institute, University of California, San Francisco, CA 94143, USA 3Present address: Institut fMr Genetik und Mikrobiologie, Universitait Miinchen, D-8000 Mulnchen 19, Germany Communicated by R.Kahmann
The Gin recombination system of phage Mu mediates inversion of the DNA sequence between two sites (gix). In addition to Gin protein and gix sites, recombination requires an enhancer bound by the host factor FIS. We analyzed mutants of Gin that function in the absence of the enhancer and FIS and mediate deletion and intermolecular fusion in addition to inversion. The linking number changes caused by inversion imply that mutant Gin alone can form the same synaptic complex and can use the same strand exchange mechanism as the complete wild-type system. However, the linking number changes also reveal that unlike wild-type Gin, mutant Gin can recombine through more than one synaptic complex and can relax DNA in the absence of synapsis. This expanded repertoire allows mutant Gin to mediate DNA rearrangements not performed by wild-type Gin. Because mutant Gin, but not wild-type Gin, unwinds gir site DNA upon binding, we postulate that FIS and the enhancer function with (-) supercoiling to promote this unwinding with wild-type Gin. The analysis of the topological changes during DNA fusion shows that both the parallel gix site configuration and the right-handed rotation of the sites during exchange of wild-type Gin are a result of the (-) supercoiling of the substrate and the number of entrapped supercoils in the synaptic complex. Key words: DNA supercoiling/FIS/recombination/ recombinational enhancer/site-specific
Introduction Site-specific recombination plays a major role in many in prokaryotic and eukaryotic cells. Examples include the resolution of intermediates in DNA transposition, the regulation of alternate gene expression, the amplification of plasmids, the integration of viral DNA into host chromosomes and the excision of proviral DNA (Cox, 1989; Landy, 1989; Stark et al., 1989a; Nash, 1990; Johnson, 1991). Site-specific recombination systems have been studied in depth biochemically, topologically and genetically, and have provided model systems for the study of the structure and function of specific nucleoprotein assemblies and of DNA supercoiling (Bliska and Cozzarelli, 1987; Boles et al.,
processes
Oxford University Press
1990; Echols, 1990; Kanaar and Cozzarelli, 1992). The bacterial DNA invertases are a particularly well characterized family of closely related site-specific recombination systems (Hatfull and Grindley, 1988; Glasgow et al., 1989; Johnson, 1991). Members include the Hin phase variation system of Salmonella typhimurium and the subject of this study, the Gin system of bacteriophage Mu. Recombination by Gin is restricted to inversion, both in vivo and in vitro (Plasterk and Van de Putte, 1984). In vivo, this leads to the alternate expression of two sets of tail fiber genes that dictate the host range of the phage (Kamp et al., 1978; Van de Putte et al., 1980). The DNA rearrangement is between two inversely oriented recombination sites, called gix. Each gix site consists of two 12 bp inversely oriented binding sites for Gin (half-sites) separated by an asymmetric 2 bp spacer called the cross-over region (Klippel et al., 1988a; Mertens et al., 1988). The sequence polarity of the cross-over region confers directionality on the gix site. Recombination requires an additional cis-acting DNA sequence called the enhancer, because it functions independently of its orientation and distance from the gix sites (Kahmann et al., 1985; Johnson and Simon, 1985). Enhancer function requires binding of the Escherichia coli host protein, FIS, at two 15 bp sites within the 60 bp enhancer sequence (Koch and Kahmann, 1986; Bruist et al., 1987). Recombination also requires a (-) supercoiled substrate DNA. The Gin reaction can be divided conveniently into two steps: juxtaposition of the recombination sites during synapsis and reciprocal double-strand exchange. A current model for Gin recombination incorporating these steps is shown in Figure 1A (Kanaar et al., 1990). In synapsis, the two Ginbound gix sites and the FIS-bound enhancer come together at a branch point in a (-) supercoiled DNA substrate. The formation of this nucleoprotein complex entraps two (-) supercoils between the gix sites. Entrapped supercoils are defined as those that would remain after nicking of the DNA but without dissociation of the synapsed recombination sites. As explained below, entrapped supercoils are an important aspect of the reaction mechanism. Strand exchange is initiated when Gin induces a staggered double-strand break in the cross-over region of each gix site. Subsequently, one pair of synapsed half-sites rotates about the other pair and religation completes inversion. The rotation is defined as right-handed because it leads to a right-handed winding of the recombined regions about each other. The topological features of the model are firmly established by the following results. The first is the topology of the products of processive recombination, in which the synaptic complex does not dissociate after one round of strand exchange. The structure of knotted DNA products from successive rounds of processive recombination demands the topological features of the model (Kanaar et al., 1990; Heichman et al., 1991). Secondly, the topology of the products generated from knotted and catenated DNA 1047
A.Klippel et al.
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Fig. 1. Ribbon diagrams of DNA illustrating changes in linking number by Gin recombination. A DNA substrate for Gin is schematically represented by a ribbon in which the edges (black and gray lines) are the complementary strands. One side of the ribbon is gray, the other white. The split-color arrows indicate the inversely oriented gix sites bound by Gin and the gray box indicates the enhancer bound by FIS. The DNA substrates for Gin need to be (-) supercoiled, but all twist and writhe not essential for synapsis or exchange are removed for clarity. A. Topological changes during DNA inversion by wild-type Gin. Synapsis of the two gix sites and the enhancer traps two (-) supercoils as shown on the left. The trapped crossings, or nodes, are indicated by -1. Recombination occurs via a double-strand break in each gix site, a 1800 right-handed rotation of one pair of half-sites relative to the other and religation to generate the product diagrammed on the right. The rotation of the DNA creates 1048
one additional (-) node, while simultaneously overtwisting both sites by a half turn (indicated by + 1/2); after deproteinization, these nodes cancel. The change in linking number (ALk) of +4 that results from recombination is solely a consequence of the change in sign of the two supercoils trapped by the synaptic complex (Cozzarelli et al., 1984). The change in sign results because inversion does not alter the overlay of the DNA segments that make up the nodes, but changes their relative orientation. The linking number of the substrate (LkS), the product (LkP) and ALk (LkP-LkS) are indicated. B. DNA inversion via a synaptic complex with no entrapped supercoils. If synapsis of the gix sites occurs without entrapment of (-) supercoils, then recombination by the same strand exchange mechanism as in A will be accompanied by a ALk of 0. FIS and the enhancer are omitted because this reaction occurs only with mutant Gin proteins that do not require these accessory factors. C. Recombination via a synaptic complex with one entrapped (-) supercoil. Entrapment of a single (-) supercoil at the time of site synapsis causes the gix sites to align in an antiparallel configuration. Because of this, an even number of 1800 rotations of the staggered breaks in the gix sites is required before complementarity in the overlap region is restored and religation can occur. Two right-handed rotations are shown, which result in a ALk of +4. In contrast to the reactions in A and B where the synapsed sites are in parallel, there is no change in DNA sequence. Consequently, the sign of the entrapped supercoil is not changed and
substrates also leads uniquely to this model (Kanaar et al., 1989). Thirdly, the model explains why DNA inversion is accompanied by a change in linking number (ALk) of +4 (Kahmann et al., 1987; Kanaar et al., 1988). The changes in Lk that are concomitant with recombination form the basis for a number of the conclusions in this paper and therefore we will discuss the properties of ALk that allow us to make deductions about the mechanism of recombination. DNA inversion changes the sign of the supercoils entrapped by synapsis. When inversion proceeds through the 'break-rotate-rejoin' strand exchange mechanism described for Gin in Figure 1A, this change is the only contribution to ALk. The DNA crossings, or nodes, introduced by the DNA motions during exchange do not contribute to ALk because they cancel. In the example shown in Figure LA, the right-handed rotation of the broken strand introduces a (-) crossing of the two gix sites and a compensating + 1/2 twist within each gix site. Therefore, if there are two entrapped (-) supercoils in the synaptic complex, the ALk accompanying inversion is +4. More than one such right-handed rotation and any number of left-handed 1800 rotations would tie a knot in all products of recombination. Because the products are unknotted under non-processive reaction conditions (Kanaar et al., 1988), strand exchange must involve a single right-handed 180° rotation. An important feature of the wild-type Gin reaction is its strict specificity for inversion. This selectivity can be explained by the requirement for a (-) supercoiled substrate and a synaptic complex containing a fixed number of entrapped (-) supercoils. We adopt the convention of viewing the synaptic complex in only one projection, shown in Figure 1, to give a unique definition to parallel (Figure 1A and B) and antiparallel (Figure IC) gix site configurations. The oriented interwound structure of (-) supercoiled DNA favors kinetically and/or thermodynamically the parallel configuration of inversely oriented sites if the number of supercoils trapped by synapsis is even, as it is for Gin (Benjamin and Cozzarelli, 1986; Craigie and Mizuuchi,
Recombination by enhancer-independent Gin mutants
1986; Boocock et al., 1987). The same directing forces favor the antiparallel configuration if the sites are directly repeated and the even number of trapped supercoils is preserved. An antiparallel configuration demands an even number of 1800 strand rotations before complementarity of the base pairs in the cross-over region is restored and ligation can occur (Figure IC); as a result, the broken strands are always rejoined to their original partners (Kanaar et al., 1990). Therefore, inversion, but not deletion, is carried out by wildtype Gin. Intermolecular fusion by Gin is precluded because (-) supercoiling does not aid a reaction between gLx sites on unlinked DNA rings (Kanaar and Cozzarelli, 1992). Therefore, understanding how recombination selectivity is achieved by Gin reduces to explaining why only a synaptic complex with two entrapped (-) supercoils is productive and what the precise roles of (-) supercoiling, FIS and the enhancer are. An important opportunity to address these questions was provided by the isolation of the novel mutants of Gin that dispensed with the need for FIS, the enhancer and (-) supercoiling and simultaneously lost the selectivity for inversion (Klippel et al., 1988b). In this report, we determined the ALk's accompanying DNA inversion and intermolecular fusion by three mutant Gin proteins and analyzed the interaction of the mutant Gin proteins with gix site DNA by footprinting and by the change in Lk induced by Gin binding. We find that inversion by mutant Gin proceeds through more than one pathway. One pathway results in a ALk of +4, as in the wild-type Gin system. This implies that mutant Gin, in the absence of FIS and the enhancer, can assemble the same branched synaptic complex and use the same strand exchange mechanism as the complete wild-type Gin system. Another inversion pathway causes a ALk of 0, indicating a second productive synaptic complex with no entrapped supercoils. The additional synaptic complexes formed by mutant Gin allow it to bypass the recombination selectivity of the wild-type Gin system. We suggest that a post-synaptic role of FIS, the enhancer and (-) supercoiling is to assist in a partial gix site unwinding that is required for initiation of strand exchange. We also suggest that mutant Gin bypasses this trio of accessory factors because the binding of mutant, but not wild-type Gin, distorts the gix site and unwinds the double helix by one-half turn and that this untwisting is necessary for exchange. In the absence of (-) supercoiling, mutant Gin recombines DNA via exchanges with either a right-handed or left-handed direction. Therefore, the exclusively right-handed exchanges with a (-) supercoiled substrate are dictated by the energetics of supercoiling.
Results Mutants of Gin that exhibit a FIS- and enhancerindependent phenotype Our experiments were carried out with three different FISindependent mutant Gin proteins (Klippel et al., 1988b). GinMV/KR has two amino acid changes compared with wild-type Gin: methionine is replaced by valine at amino acid position 114 and lysine is replaced by arginine at position 162. The latter mutation is silent and the FISindependent phenotype is caused solely by the mutation at position 114. GinMV1 14 carries just the substitution at position 114. GinHY106 has a histidine to tyrosine change
Fig. 2. Supercoil relaxation by mutant Gin is uncoupled from recombination. Supercoiled DNA (2.5 jg) with (pAZ-L,B, lanes 1-5) or without (pTZ19U, lanes 6-10) a gix site and enhancer was incubated at 37°C for 30 min in the presence of 100 ng of wild-type Gin (WT), 100 ng GinMV114 (MV) and 20 ng FIS as indicated. Onethird of the DNA was electrophoresed through a 1% agarose gel containing TBE and 0.5 jsg/ml chloroquine at 2 V/cm for 24 h. The ethidium bromide-stained gel is shown. The position of the nicked open circular form of each plasmid is indicated (oc). The bands ahead of the oc position are (-) supercoiled topoisomers.
at position 106. We will refer collectively to the FISindependent Gin proteins as mutant Gin. Uncoupling of recombination and relaxation activities of mutant Gin Unlike Tn3 resolvase and the X integrase, the wild-type Gin system does not promote extensive relaxation of its (-) supercoiled substrate (Kanaar et al., 1986). The relaxation detected is coupled with recombination and changes Lk by +4 (see Figure 1A). Thus, relaxation by Gin has the same requirements as recombination: two gix sites, the enhancer and FIS, suggesting that any DNA breakage and reunion by Gin, whether for strand exchange or relaxation, requires a complete synaptic complex. We found that the Gin mutants lacked this constraint and promoted extensive relaxation uncoupled from recombination (Figure 2). This was shown most clearly with a substrate containing a single gix site, which precludes intramolecular synapsis. GinMV1 14 relaxed supercoiled pAZ-L,B DNA, containing only a single gix site and the enhancer, with high efficiency both in the presence and absence of FIS (Figure 2, lanes 4 and 5). Similar results were obtained with GinMV/KR and GinHY 106 (data not shown). This activity of mutant Gin is in sharp contrast to that of wild-type Gin, which did not relax this substrate in either the presence or absence of FIS (Figure 2, lanes 2 and 3). The Gin mutants are not acting simply as non-specific topoisomerases because they did not relax DNA without a gix site (Figure 2, lanes 9 and 10). In a control, we showed that under the conditions used for relaxation, both wild-type and mutant Gin efficiently recombined pAZ-IR2, which contains two inversely oriented gix sites and the enhancer (data not shown). We conclude that mutant Gin can break gLx site DNA, pass a DNA strand through the break and rejoin the DNA without FIS, the enhancer or intramolecular synapsis to another gix site.
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Change in linking number associated with inversion by mutant Gin To determine the topological structure of the synaptic complex and the mechanism of strand exchange, we measured the ALk accompanying inversion by mutant Gin. It is helpful to consider first the ALks that can accompany inversion through a double-strand break and rotation mechanism. Figure IA shows the ALk during inversion by wild-type Gin. Two (-) supercoils are trapped by synapsis of the sites and it is the change in their sign that causes the ALk of +4. The changes in Lk introduced by the DNA rotation cancel. Thus, if no supercoils are trapped by synapsis, then ALk equals 0 (Figure 1B). For simplicity, we will designate synaptic complexes by the number and sign of the entrapped supercoils. The inversely oriented gix sites are in the parallel configuration in the 0 and -2 synaptic complexes, and thus these synaptic complexes are competent for inversion. The -1 complex (Figure 1C) and other complexes with an odd number of entrapped supercoils, have an antiparallel configuration and therefore do not lead to inversion. The plasmid substrate used to measure the ALk accompanying inversion, pIR2, contains two inversely oriented gix sites and the enhancer. Three different topoisomers from a native Lk distribution were prepared and are called A, B and C in order of decreasing (-) supercoiling. They were incubated with GinMV/KR in the absence or presence of FIS under conditions that minimized processive strand exchange and the resultant complex knotting that complicates the identification of the reaction products (Kanaar et al., 1990). Reactions containing wild-type Gin and FIS served as controls. Reactions were stopped after only 3 min to minimize the uncoupled DNA relaxation activity of mutant Gin. Under these conditions, the total amount of recombination for mutant Gin was 20% in the presence of FIS and 30% in its absence, as determined by restriction enzyme digestion of the reaction products. The undigested recombination products were separated on a chloroquinecontaining agarose gel (Figure 3A). For the wild-type Gin plus FIS reaction, recombination was associated with a ALk of +4 (Figure 3A, lanes 2, 6 and 10), as expected. GinMV/KR without FIS promoted a more extensive relaxation of all three starting topoisomers and products with a ALk of +1, +2, +3, +4 and +5 were readily visible (Figure 3A, lanes 4, 8 and 12). FIS inhibited relaxation somewhat and enhanced the relative amount of material in the +4 topoisomer (Figure 3A, lanes 3, 7 and 11). In order to interpret these results, we discriminated between the ALk caused by inversion and that caused by relaxation uncoupled from recombination. This was done by measuring the extent of inversion for each product topoisomer by restriction enzyme digestion. Relaxation, unlike one round of inversion, does not change nucleotide sequence. We used two-dimensional gel electrophoresis to resolve the complex array of products resulting from concurrent recombination and relaxation. The extent of inversion for the products of topoisomer A are shown in Figure 3B. In the reactions by GinMV/KR and FIS, the topoisomers with a ALk of +4 were mostly recombinant. Topoisomer +5 contained a lower ratio of recombinant to parental DNA and topoisomers 0, + 1, +2 and +3 contained only trace amounts of product. Taking into account the relative amount of the topoisomers and the extent of inversion
1050
for each topoisomer, we calculate that -90% of the recombinants had a ALk of +4 or +5 and 10% had a ALk of 0 to +3. We interpret the pattern as follows. The major product of recombination has a ALk of +4 and the +5 topoisomer is generated by relaxation of the +4 topoisomer. Rare recombinants have a ALk of 0 and the recombinants in topoisomers +1 to + 3 arise from relaxation of the 0 recombinant topoisomer. We suggest that in the presence of FIS and the enhancer, mutant Gin recombines by a mechanism essentially identical to that of wild-type Gin. In the reaction mixtures containing mutant Gin without FIS, most of the recombination was again associated with the ALk +4 and +5 topoisomers (Figure 3B). This suggests that mutant Gin, in the absence of FIS and the enhancer, can recombine through the same or topologically equivalent, -2 synaptic complex and through the same strand exchange mechanism as the wild-type Gin system. Thus, FIS and the enhancer do not make essential contributions to the standard synaptic complex and mechanism of exchange. A substantial amount of recombination is also associated with the ALk 0, + 1, +2 and +3 topoisomers in the reactions without FIS (Figure 3B). We interpret this as due to recombination with ALk of 0, through the 0 synaptic complex (see Figure IB), followed by uncoupled relaxation in steps of 1 to generate the +1, +2 and + 3 topoisomers. Based on the relative amount of product topoisomers and the percentage of recombinants in each topoisomer, we estimate that 60% of the recombinants were generated through the -2 synaptic complex and 40% through the 0 complex. We conclude that mutant Gin can recombine through at least two pathways. One pathway is identical to that of wildtype Gin and proceeds through the -2 synaptic complex. The other, unique to mutant Gin, proceeds through the 0 synaptic complex. The two (-) supercoils entrapped in the synaptic complex that are essential for recombination in the wild-type Gin system are dispensable for mutant Gin.
Changes in DNA topology during intermolecular recombination: directionality of strand exchange In order to gain information about the mechanism of a reaction that is unique to mutant Gin, we measured the ALk and the extent of catenation associated with intermolecular recombination. In Figure 4 we consider the most likely mechanisms for intermolecular recombination. As with intramolecular reactions, a parallel gix site configuration can lead to nucleotide rearrangement; i.e. fusion (Figure 4A). If two identical plasmids are fused without net change in twist and writhe, the Lk of the dimeric circular product will be twice that of the starting monomeric plasmids. The difference from this doubled value of Lk we define as the ALk of fusion. Strand exchange via right-handed rotation yields a ALk for fusion of +2; left-handed rotation leads to a ALk of -2. If the gix sites are aligned instead in the antiparallel configuration, an even number of 1800 strand rotations is required for strand exchange (Figure 4B). In this case, strand rotation in either direction forms a singly linked catenane with a change in Lk, but not in DNA sequence. We first determined the ratio of circular dimers and singly linked catenanes among products of intermolecular recombination by GinMV/KR (Figure 5). We used a relaxed topoisomer of pAZ-Lj, because relaxed substrates gave a higher yield of intermolecular recombination products than supercoiled substrates. The data shown in Figure 5 are for
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Fig. 3. The change in linking number accompanying inversion by mutant Gin is +4 or 0. A. Linking number change during Gin inversion. As indicated, the reaction mixtures contained one of three purified topoisomers of pIR2: A, B or C and 50 ng wild-type Gin (WT), 40 ng Gin MV/KR (MV/KR) and 25 ng FIS. The products were separated by electrophoresis at 2 V/cm for 52 h through a 1.3% agarose gel containing TBE and 6 /Ag/ml chloroquine. The DNA was transferred to Nytran and probed with 32P-labeled pIR2 DNA. The resulting autoradiogram is shown. The marker lane, M, shows a distribution of (-) supercoiled topoisomers of pIR2 DNA. The substrate topoisomers had a higher electrophoretic mobility than the product topoisomers, because they contained more (-) supercoils. The position of the product topoisomer for which Lk increased by four is indicated by an open arrowhead. Nicked plasmid DNA is the band at the top of the photograph. In lane 2, the minor band running just ahead of the major product is probably a product with a ALk of +4 from the contaminating substrate topoisomer. A minor product is not present in lanes 6 and 10, for which the substrates were topologically more homogeneous. B. Restriction endonuclease analysis of product topoisomers. Product DNA from reaction mixtures containing topoisomer A and GinMV/KR with and without FIS were separated by electrophoresis in the first dimension as described in A. The gels were then soaked in TAE with 0.03% SDS and run in a second dimension for 30 h at 2 V/cm to increase the resolution of the products. After ethidium bromide staining, topoisomers 0 to +5 and nicked open circular DNA (oc) were isolated, digested with Pvull and BamHI endonucleases and subjected to electrophoresis through a 1 % TBE-agarose gel. The gel was blotted and probed and an autoradiogram of the diagnostic region of the gel is shown. The restriction digests of the reactions without FIS were placed below the corresponding lanes for the reactions in the presence of FIS. The positions of the 1.0 kb parental (P) and 1.2 kb recombinant (R) DNA fragments are indicated. Lane M' contains markers of parental and recombinant restriction digests of pIR2.
a topoisomer containing a single (+) supercoil. The concentration of DNA was increased from 10-75 jg/ml to
promote intermolecular reactions. Three-quarters of the reaction products were nicked with DNAase I and separated by high resolution agarose gel electrophoresis (Figure SA). The total amount of intermolecular recombination was 5 %. About two-thirds of the products were dimeric circles,
Recombination by
GGin mutants
indicative of recombination through parallel gix sites, and one-third were singly linked catenanes, indicative of recombination through antiparallel gix sites. Therefore, the asymmetric 2 bp cross-over sequence and any sequences outside of the gix site are not important determinants of site configuration in recombination. We then measured the ALk of the dimeric fusion products using the remaining one-quarter of the DNA from the above reactions to determine if there was an intrinsic direction to site rotation during strand exchange. The use of a relaxed topoisomer removed any directing effect of (-) supercoiling, which biases the reaction towards a positive ALk. The DNA was resolved by electrophoresis through a chloroquinecontaining agarose gel (Figure SB) and the Lk of the reaction products was determined by reference to overlapping topoisomer ladders (Figure 5B, lanes 5-8). Under the gel electrophoretic conditions used, the relaxed monomer substrate topoisomer had six (+) supercoils. If a circular dimer formed with no change in Lk, it would run at position + 12. The two major dimeric products migrated at positions + 10 and + 14, which correspond to ALks of -2 and +2, respectively (Figure SB, lane 2). This indicates that mutant Gin can mediate strand exchange by right-handed or lefthanded rotations. The ratio of the + 10 and + 14 products depended on the supercoiling density of the substrate in the expected way. For the topoisomer substrate containing a single (+) supercoil, the + 10 product band was a little more intense than the +14 band (Figure SB, lane 2). When slightly (-) supercoiled topoisomers were used as substrate, the predominant product was now the +14 topoisomer product, indicating a ALk of +2 (data not shown). At high Gin concentrations, uncoupled topoisomerase activity relaxed the products to a distribution about position + 12 and the substrate to about position +6 (Figure SB, lane 4). The simplest interpretation of the data is that mutant Gin performs intermolecular recombination through the 0 synaptic complex containing the gix sites in either the parallel or antiparallel configuration and that (-) supercoiling, not Gin itself, imposes a directionality on strand exchange. Binding of mutant Gin recombinases to the gix site We sought next to define the differences in the way mutant Gin interacts with gix site DNA that allow it to be active for recombination in the absence of accessory factors. We tested whether wild-type and mutant Gin differed at the level of DNA binding by footprinting with the iron chelate of methidium propyl -EDTA [MPE * Fe(ll)], a DNA cleaving agent that intercalates via the minor groove. End-labeled DNA fragments containing a single gix site were treated with MPE * Fe(ll) after preincubation with increasing amounts of wild-type and mutant Gin. The footprint patterns are shown in Figure 6. Wild-type Gin protected both half-sites of the gix site, but not the 2 bp cross-over region (lanes 3-5). Gin mutants GinMV/KR and GinHY106 also protected the halfsites Oanes 6-11) and the binding affinity and cooperativity were about the same as for the wild-type protein. However, in contrast to wild-type Gin, mutant Gin cleaved one of the two strands of the gix site DNA at the cross-over region (Figure 6, tailed arrow). This conclusion is based on additional experiments showing the generation of the same intense band in the absence of MPE * Fe(II), the mapping of the break to the expected nucleotide in the cross-over region and the demonstration that the nucleotide at the break was
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Fig. 4. Topological changes during intermolecular recombination by mutant Gin. Shown are ribbon diagrams representing relaxed DNA substrates for Gin. Symbols are the same as in Figure 1. A. The gix sites are in the parallel configuration in the intermolecular synaptic complex depicted on the left. The same strand exchange events shown in Figure 1, a double-strand break in the gix sites followed by a 1800 right-handed rotation (1800, RH) and religation, will result in a ALk of +2 (top). A left-handed 1800 rotation of the broken gix sites (1800, LH) will result in a ALk of -2 (bottom). In both cases, the two substrate plasmids fuse to form a dimer. B. The gix sites are in the antiparallel configuration in the intermolecular synaptic complex on the left. Because of this configuration, an even number of 1800 rotations is required before religation and therefore the parental nucleotide sequence is preserved. Recombination through two 1800 right-handed rotations (2 x 1800, RH; top) yields a catenane linked by two (+) nodes (Ca = +2). Each plasmid ring of the catenane contains an additional (+) supercoil compared to the substrate plasmids; LkP = +1 x2 = +2. Two left-handed rotations (I x 1800, LH; bottom) link the plasmids by two (-) nodes (Ca = -2) and each plasmid gains a (-) supercoil (LkP = -lx2 = -2). Ca is 0 for the unlinked DNA rings.
covalently linked to Gin protein (not shown). This result, like the uncoupled relaxation activity of mutant Gin, implies that mutant Gin can cleave a single gix site without the assistance of FIS, the enhancer, supercoiling or synapsis. Besides cleavage of the gix site, there was a difference in the extent of the wild-type and the mutant Gin footprints. Wild-type Gin protected all but the 2 bp in the cross-over region of the gix site. However, with mutant Gin bound, this unprotected region was enlarged to 4-5 bp (Figure 6, lanes 6- 11; arrowhead), but is partly obscured by the mutant Gin cleavage product. Therefore, binding of mutant Gin altered the structure of the DNA such that the accessibility of MPE Fe(II) to the minor groove was increased in the region of strand exchange. Binding of mutant Gin at the gix site induces localized unwinding The footprinting data suggested that binding of mutant Gin distorts the DNA double helix at the cross-over region in the gix site. We investigated whether this distortion was an actual unwinding of the double helix. Singly nicked pAZLo, a plasmid containing a single gix site and the enhancer, was incubated with increasing amounts of wild-type Gin or GinMV1 14 in the presence or absence of FIS. The nick was subsequently sealed with DNA ligase to fix any change in twist or writhe of the DNA caused by protein binding. The resulting distribution of topoisomers was analyzed by
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electrophoresis through a chloroquine-containing agarose gel. Under these electrophoresis conditions, the topoisomers contain (+) supercoils. Binding of the wild-type Gin resulted in no shift in the distribution of topoisomers (Figure 7, lanes
2-5). FIS also had no effect, either alone or in the presence of wild-type or mutant Gin. In contrast, GinMVl 14 binding did result in a slower migrating and therefore more (-) topoisomer distribution (Figure 7, lanes 6-9). The direction of the topological change was confirmed by analyzing the DNA on gels containing different concentrations of chloroquine (not shown). The extent of unwinding was quantified. GinMV1 14 binding resulted in an induced ALk of about -0.5 in a single gix site plasmid. For pAZ-IR2, containing two gix sites, a change of - 1.1 was found (data not shown). Results with plasmids pL (single gix site) and pIR2 (two gix sites) were similar to those with pAZ-L,B and pAZ-IR2, respectively. Mutants GinMV/KR and GinHY 106 caused unwinding similar to GinMVl 14 (data not shown). The simplest interpretation of the results is that binding of mutant Gin caused a localized unwinding of about half a helical turn per gix site. This is equivalent to an unwound region of 5 bp.
Discussion We studied the mechanism of strand exchange, the structure of the synaptic complex and the DNA binding by mutant Gin
Recombination by enhancer-independent Gin mutants
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Fig. 5. Changes in DNA topology caused by intermolecular recombination by mutant Gin. A. Products of intermolecular recombination. A relaxed topoisomer (5 Ag/ml) of pAZ-L,B was incubated with 0, 6, 9 and 12 gg/ml Gin MV/KR (lanes 1-4) in a 40 1.1 reaction volume for 60 min. To determine the amount of circular dimeric and catenated products, three-quarters of the DNA was nicked and resolved by electrophoresis through a 1% agarose gel containing TAE and 0.03% SDS. A photograph of the ethidium-stained gel is shown. Lane M contains nicked open circular (oc) dimer, supercoiled (sc) dimer and knotted DNA markers containing 3, 5, 7 and 9 nodes. The positions of linear (lin) and nicked open circular (oc) monomer and the singly-linked catenated product are also indicated. B. Changes in linking number during intermolecular fusion. One-quarter of the unnicked products of the reactions in A were separated by electrophoresis through a 1% agarose gel containing TBE and 0.5 ig/ml chloroquine (lanes 1-4). DNA in the gel was transferred to Nytran and probed with 32P-labeled pAZ-L,B DNA. An autoradiogram is shown. Under the electrophoretic conditions employed, the starting topoisomer contained six (+) supercoils (indicated on left). All topoisomers are indexed relative to this number and the fusion dimeric product with no change in Lk is accordingly indexed at + 12. The major dimeric products are topoisomers + 10 and + 14 (lane 2), which therefore have gained, respectively, two (-) and two (+) supercoils by recombination. Overlapping distributions of marker (-) topoisomers (lanes 5-8) were generated by incubating a mixture of monomeric and dimeric pAZ-L3 with calf thymus topoisomerase I in the presence of increasing concentrations of ethidium bromide. The concentrations of ethidium bromide used were 0, 1, 2 and 2.5 ag/ml, respectively (lanes 5-8).
proteins that recombine DNA in the absence of FIS, the enhancer and (-) supercoiling. Our findings have implications for recombination site synapsis, strand exchange and the role of FIS and the enhancer.
Synapsis of recombination sites The experiment presented in Figure 3 shows that inversion by mutant Gin in the absence of FIS can be accompanied by a ALk of +4, just as it is in the wild-type Gin system (Kahmann et al., 1987; Kanaar et al., 1988). This result implies that no critical Gin-FIS contacts are required to set up a synaptic complex containing two Gin-bound gix sites at a branch point in (-) supercoiled DNA (Figure 1A). All necessary components to assemble this nucleoprotein complex at a branch point must be provided solely by Gin and the structure of (-) supercoiled DNA. Gin-FIS contacts may play a role at a later stage of the reaction, but these cannot be essential for gix site synapsis. Recombination by the wild-type Gin system proceeds
exclusively through this -2 synaptic complex (Kanaar et al., 1988, 1989, 1990). Mutant Gin does not have this constraint. Inversion by mutant Gin is associated with a ALk of either +4 or 0 (Figure 3). The latter value implies that mutant Gin can invert DNA through an alternative synaptic complex entrapping no (-) supercoils between the gix sites (Figure 1B). The alternative synaptic complex formed by mutant Gin allows it to escape the recombination selectivity of wild-type Gin. The 0 synaptic complex is uniquely well suited for intermolecular recombination because the lack of site intertwining eliminates the need for compensatory supercoiling outside of the synaptic complex. The analysis of the topology of knotted products from processive recombination proves that mutant Gin also mediates reactions through a -1 synaptic complex (N.J.Crisona, R.Kanaar, A.Klippel, R.Kahmann and N.R.Cozzarelli, unpublished results). With directly repeated gix sites, the -1 synaptic complex has a parallel site configuration, which allows 1053
.
A.Klippel et a!.
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Fig. 6. Protection of gix site DNA from chemical nuclease by Gin proteins. Wild-type (WT), MV/KR and HY106 Gin proteins were incubated with DNA containing a gix site. The amount of Gin used was 12 ng (lanes 3, 6 and 9), 6 ng (lanes 4, 7 and 11) and 3 ng (lanes 5, 8 and 12). The gix site was present on a 630 bp HincH-ScaI fragment from plasmid pIRsym2 that was labeled at the 5'-end of the ScaI site with 32p. The binding mixtures were then treated with MPE Fe(ll) and the products were resolved on a 6% polyacrylamide gel under denaturing conditions. Solid bars at the right mark the protected half-sites in the gix site. The tailed arrow indicates the site of cleavage in the gix site by the mutant Gin proteins. Because Gin is covalently attached to the revealed 5'-phosphoryl group, the labeled strand is protein-free. The arrowhead marks the lower border of the region that is accessible to MPE- Fe(II) attack when mutant, but no wild-type Gin is bound to the gix site. Marker lanes are: lane 1, Gand A-specific Maxam and Gilbert sequencing reaction; lane 2, no protein.
deletions. By forming 0, -1 and -2 complexes, mutant Gin carry out the complete spectrum of sequence rearrangements of site-specific recombination systems. The analysis of mutant Gin reactions also allowed us to determine whether the parallel or antiparallel configuration of the gix sites is favored in the synaptic complex. Figure 5A shows that intermolecular recombination by mutant Gin yields comparable amounts of dimeric circles and singly linked catenanes. The dimeric circles are the result of a parallel configuration of the gix sites in the synaptic complex and the singly linked catenanes are indicative of an antiparallel configuration (Figure 4). Thus, there is no pronounced intrinsic favoring of a particular site configuration by Gin. Evidently, the formation of the complex is dictated by the dyadic Gin binding sites and not 1054 can
type (WT) Gin or GinMV114 in the absence (-) or presence (+) of 4 ng FIS. T4 DNA ligase and ATP were added after 5 min and the incubation was continued for 30 min. The resulting topoisomers were resolved on a 1% TBE-agarose gel containing 0.5 ,ug/ml chloroquine. Electrophoresis was at 2 V/cm for 24 h and the ethidium bromidestained gel is shown. Because the DNA is (+) supercoiled under the electrophoresis conditions, introduction of a (-) supercoil retards mobility. Lane M contains marker (BRL) 2036 and 3054 bp linear DNAs. Lane M' contains substrate DNA to which no DNA ligase was added. The nicked open circular (oc) and linear (in) forms of the plasmid DNA migrate slower than the topoisomer distributions.
the asymmetric cross-over sequence. For intramolecular recombination by wild-type Gin, site configuration is instead specified by the structure of the (-) supercoiled substrate and the requirement for the -2 synaptic complex. Supercoiling greatly favors the parallel configuration of inversely oriented gix sites in a -2 synaptic complex. Conversely, the antiparallel configuration is favored for a -2 synaptic complex on a substrate containing directly repeated gix sites (Kanaar and Cozzarelli, 1992). Directionality of strand exchange For the wild-type Gin system, recombination within a (-) supercoiled substrate always proceeds through a right-handed rotation of the broken gix sites around each other (Kanaar et al., 1988, 1990). The independence of mutant Gin from (-) supercoiling allowed us to test whether the direction of strand rotation is dictated by Gin or by the structure of (-) supercoiled DNA. Intermolecular fusion through the 0 synaptic complex with the gix sites in the parallel configuration will produce a dimeric circle. The ALk accompanying this reaction is +2 if strand rotation is righthanded and -2 if it is left-handed (Figure 4A). We found that products with a ALk of +2 and -2 were formed equally with relaxed substrates (Figure 5B). With slightly (+) supercoiled topoisomers left-handed strand rotations predominated, whereas with slightly (-) supercoiled topoisomers right-handed strand rotation predominated. Thus, directionality of strand exchange in the Gin system is dictated by (-) supercoiling. This seems to be a general feature for site-specific recombination, because a similar
Recombination by enhancer-independent Gin mutants
conclusion has been made for phage X integrase, phage P1 Cre and Tn3 resolvase (Nash and Poilock, 1983; Abremski et al., 1986; Stark et al., 1989b). The mechanism by which (-) supercoiling directs strand exchange can be rationalized on energetic grounds. During right-handed strand exchange, the gix sites cross once to form a (-) writhe node, while the DNA double helix in each site overtwists by one-half turn, creating two + 1/2 nodes (see Figure 4A). A left-handed rotation causes a (+) writhe node and two -1/2 nodes in each gix site. As long as the synaptic complex stays intact, the writhe nodes should be energetically similar for strand rotations in either direction (Kanaar and Cozzarelli, 1992). However, (-) supercoiling energetically favors the (+) twist nodes put in by right-handed rotations and disfavors the (-) twist nodes resulting from left-handed rotations. Thus, (-) supercoiling imposes a directionality on strand exchange by greatly promoting right-handed strand rotations over left-handed strand rotations. The recombination proteins function solely to induce DNA breakage and reunion and to limit the number of strand rotations. Model for FIS and enhancer action The ALk of +4 during DNA inversion by mutant Gin in the absence of FIS implies that FIS and the enhancer have no essential role in gix site synapsis and strand exchange by the mutant protein. Thus, it is unlikely that FIS and the enhancer are intimately involved in strand rotation during recombination by the wild-type system. Instead, we suggest that FIS and the enhancer act after synapsis but before strand exchange. From our observations on the mechanism of mutant Gin recombination, we derived the following model for FIS and enhancer action. We propose that wild-type Gin, by itself, is unable to initiate breakage of strands until FIS and the enhancer become part of the synaptic complex. FIS and the enhancer induce some conformational change in the Gin-gix supercoiled complex that initiates exchange. The mutant Gin-gix complex is already in the activated conformation and thus dispenses with FIS, the enhancer and (-) supercoiling. We surmise that the change in the Gin-gix complex brought about by FIS and the enhancer is the partial unwinding of the gix site. It is reasonable to assume that this unwinding is caused by the relative rotation of the two Gin-bound half-sites of the gix site. Mechanistically this may be accomplished by reorganizing the contacts between the monomers that form the Gin dimer bound to the gix site. The mutations in Gin that cause the FIS-independent phenotype are located at amino acid positions 106 and 114. When Gin is modeled on the crystal structure of the catalytic domain of 'y5 resolvase, these positions are located in an a helix that constitutes the solution dimer interface (Hughes et al., 1990; Sanderson et al., 1990). Amino acid changes at that interface could change the position of the monomers with respect to each other, and as a result the bound half-sites are rotated relative to each other. Local unwinding could be a prerequisite for recombination by disrupting the base pairing in the cross-over region of the parental gix site and aiding the juxtaposition of the complementary recombinant strands in the ligation step. The crystal structure of the catalytic domain of 'y6 resolvase placed the two active site serine residues too far apart to make a 2 bp staggered break in the
B-type double helix (Sanderson et al., 1990). Bending of res site DNA combined with a partial unwinding of the site may help position the serines more favorably (Hatfull et al., 1987; Salvo and Grindley, 1988; P.Rice and T.Steitz, personal communication). The model explains the high relaxation activity of mutant Gin compared with wild-type Gin. From the observation that mutant Gin can relax DNA containing a single gix site we infer that it does not require the formation of a complete synaptic complex in order to break and rejoin the strands. Figure 6 shows that mutant Gin can also nick one of the strands of DNA with a single gix site. In a (-) supercoiled DNA, this will lead to relaxation by swiveling of the strands around each other before religation. Relaxation activity is not expected for wild-type Gin because it requires the assembly of a complete synaptic complex before strands can be broken. The model also explains the loss of recombination selectivity by mutant Gin. Mutant Gin loses selectivity because it can independently induce structural alterations in the giX site. This allows mutant Gin to dispense with the need for FIS, the enhancer and (-) supercoiling, and to recombine through multiple synaptic complexes. The unique arrangement of the Gin-bound gix sites at a branch point in the -2 synaptic complex allows FIS-bound enhancer to readily interwind with the gLx sites, thereby rendering wildtype Gin recombination competent only in the -2 synaptic complex (Kanaar and Cozzareili, 1992). Specificity for inversion seems to have provided the selective advantage that guided the evolution of the relatively elaborate wild-type system. The finding that FIS and the enhancer are not required for gix site synapsis fits nicely with the proposal that the enhancer functions during processive recombination in a hitand-run fashion, whereby the enhancer does not remain in the synaptic complex during the multiple rounds of strand exchange (Kanaar et al., 1990). The bending of DNA attendant to the binding of Gin may help localize the synaptic complex to a branch point in supercoiled DNA (Kanaar and Cozzarelli, 1992), even in the absence of FIS and the enhancer. Recent experiments imply that the enhancer required for Mu transposition also operates through a transient interaction with the synapsed transposon ends (Surette and Chaconas, 1992). The presence of the enhancer is required at an early stage of Mu transposition, but it is dispensable for strand cleavage. Our observation that a site-specific DNA binding protein can unwind DNA upon interaction with a ligand has precedents. In E.coli, the MerR protein is a repressor of genes involved in detoxification of the cell upon exposure to Hg(II). MerR binds between the -10 and -35 elements of the promoter it represses. MerR bound by Hg(lI) unwinds its DNA binding site, probably to phase properly the -10 and -35 elements of the promoter (Ansari et al., 1992). The MerR binding site has a modular structure like the gix site; it consists of two half-sites separated by 4 bp. The unwinding of the MerR binding site by MerR-Hg(II) correlates with increased sensitivity of the DNA between the half-sites to MPE Fe(II) (Frantz and O'Halloran, 1990), just as we found for mutant Gin binding to gix (Figure 6). Mutants of MerR that unwind DNA in the absence of Hg(II) also activate transcription in the absence of the metal. These mutations are not in the DNA binding domain and are presumably in the dimerization domain of the protein. 1055
A.Klippel et al.
Control of Gin recombination at the level of DNA
unwinding has a parallel in other DNA transactions that are mediated by specialized nucleoprotein structures. In transcription, initiation requires formation of an open promoter -RNA polymerase complex. The same theme is found in DNA replication. In several cases, initiation of replication is regulated at the level of DNA unwinding by origin binding proteins (Bramhill and Kornberg, 1988; Echols, 1990; Dean and Hurwitz, 1991). Materials and methods Enzymes Wild-type Gin and FIS were purified as previously described by Koch and Kahmann (1986), Mertens et al. (1986) and Kanaar et al. (1988). Mutant Gin proteins GinMV/KR, GinMV114 and GinHY106 were purified essentially by the procedure described for wild-type Gin. Calf thymus topoisomerase I, restriction endonucleases, T4 DNA ligase and other enzymes used for the construction of DNA substrates were from commercial sources.
Reactions Recombination reactions: Gin recombination reactions were performed at 37°C. The reaction mixtures contained 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl and 10-75 jtg/mi DNA. The amounts of Gin protein and FIS used in each reaction and the incubation time are indicated in the figure legends. Reactions were terminated by the addition of SDS to 0.5%. MPE Fe(ul) footprinting reactions: 1.2 mM of methidium propyl-EDTA (MPE), provided by P.Dervan, was mixed on ice with an equal volume of 1.2 mM Fe(NH4)2(SO4)2 and diluted 5-fold with H20. DNA cleavage was induced by adding 2.5 Al of this MPE Fe(II) solution and 2.5 1,u of 10 mM DTT to 40 fmol (50 000 c.p.m.) of a 32p 5'-end labeled gix site DNA fragment bound by the indicated amounts of Gin in 15 ftl of standard reaction buffer. After 7 min at 25°C, DNA cleavage was stopped by the addition of 1.5 ,ul of 50 mM bathophenanthroline disulphonate. The DNA was extracted with phenol-chloroform (1:1, v/v), precipitated with ethanol in the presence of 1 jtg of tRNA and lyophilized. The cleavage products were incubated for 2 min at 100°C in formamide loading buffer and 10 000 c.p.m. of each reaction was analyzed by electrophoresis through a 6% denaturing polyacylamide gel and autoradiography. DNA unwinding reactions: singly-nicked DNA substrates were incubated under standard reaction conditions with the indicated amounts of Gin and FIS at 37°C. After 5 min T4 DNA ligase and ATP were added and the incubation was continued for 30 min. For reactions containing DNA substrates with inversely oriented gLx sites, recombination was monitored in parallel by restriction enzyme digestion. The ALk of the DNA induced by protein binding was determined as described by Dean and Hurwitz (1991). DNA substrates The substrates used in this study were pIR2, pL3, pAZ-IR2, pAZ-L( and pIRsym2. Plasmid pIR2 (3.9 kb) contains two inversely oriented gix sites separated by 0.3 kb and the enhancer at wild-type distance from gixR, a center-to-center separation of 119 bp (Klippel et al., 1988a). pL3 (3.6 kb) contains a single gix site (gixR) and the enhancer at wild-type distance from gixR. Plasmids pAZ-IR2 (2.7 kb) and pAZ-L3 (2.4 kb) are similar to pIR2 and pL,3, respectively, except that the shorter pAZ vector (kindly provided by U.Guenthert) replaced pBR322. Plasmid pIRsym2 has been described by Mertens et al. (1988). Topoisomers of substrate DNA were separated by agarose gel electrophoresis and purified by electroelution (Kanaar et al., 1988). The eluted DNA was extracted with phenol and chloroform, precipitated with ethanol and resuspended in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. DNA substrates were relaxed by incubation with calf thymus topoisomerase I under Gin reaction conditions. To obtain singly-nicked open circular DNA substrates, 50 /g of DNA was incubated for 30 min at 25°C in Gin reaction buffer containing 200 ,tg/ml BSA, 125 ,ug/ml ethidium bromide and 10 jig/ml DNAase I.
Gel electrophoresis Agarose gel electrophoresis of DNA was carried out as described in the figure legends. The electrophoresis buffer was either TBE [89 mM Tris-borate (pH 8.3) and 2.5 mM EDTA)] or TAE [80 mM Tris-acetate (pH 7.5), 5 mM sodium acetate and 1 mM EDTA]. Gels were soaked sequentially in ethidium bromide (0.5 Ag/ml) and electrophoresis buffer for
1056
60 min periods. When chloroquine or SDS was present during electrophoresis, the gel was first soaked in buffer. Photography used ultraviolet illumination (254 nm) and Polaroid type 655 or type 55 film. Quantification was done by scanning photographic negatives or autoradiograms with a Hoefer Scientific Instruments GS300 densitometer coupled to a Macintosh computer.
Southern blotting The DNA in an agarose gel was denatured in 0.4 M NaOH and 0.6 M NaCl for 60 min and then transferred to a Nytran filter (Schleicher and Schuell) overnight. The filters were neutralized in 0.5 M Tris-HCl (pH 7.4) and 1.0 M NaCl for 30 min and exposed to 120 mJ/cm2 ultraviolet light (254 nm). Hybridization with 32P-labeled DNA was performed in 0.5 M NaH2PO4 (pH 7.0) and 7% SDS at 65°C for 16 h. Filters were washed twice with 0.1 M NaH2PO4 (pH 7.0) and 1% SDS and twice with 0.04 M NaH2PO4 (pH 7.0) and 1 % SDS. Autoradiography was performed using Kodak XAR-5 film. Plasmid DNA was labeled with [32P]dCTP using a multiprime DNA labeling system from Amersham.
Acknowledgements We are indebted to C.Koch for his help and advice in purifying the FISindependent Gin proteins and for providing purified GinMV1 14. This work was supported by a grant from the NIH to N.R.C. and by a grant from the Deutsche Forschungsgemeinschaft to R.Kahmann. R.Kanaar is a fellow of The Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been aided by a grant from The Jane Coffin Childs Memorial Fund for Medical Research.
References Abremski,K., Frommer,B. and Hoess,R.H. (1986) J. Mol. Biol., 192, 17-26. Ansari,A.Z., Chael,M.L. and O'Halloran,T.V. (1992) Nature, 355, 87-89. Benjamin,H.W. and Cozzarelli,N.R. (1986) Proc. RobertA. Welch Found. Conf Chem. Res., 29, 107-126. Bliska,J.B. and Cozzarelli,N.R. (1987) J. Mol. Biol., 194, 205-218. Boles,T.C., White,J.H. and Cozzarelli,N.R. (1990) J. Mol. Biol., 213, 931-951. Boocock,M.R., Brown,J.L. and Sherratt,D.J. (1987) UCLA Symp. Mol. Cell. Biol., new series 47, 703 -718. Bramhill,D. and Kornberg,A. (1988) Cell, 52, 743-755. Bruist,M.F., Glasgow,A.C., Johnson,R.C. and Simon,M.I. (1987) Genes Dev., 1, 762-772. Cox,M.M. (1989) In Berg,D.E. and Howe,M.M. (eds), Mobile DNA. American Society for Microbiology, Washington, DC, pp. 661-670. Cozzarelli,N.R., Krasnow,M.A., Gerrard,S.P. and White,J.H. (1984) Cold Spring Harbor Symp. Quant. Biol., 49, 383-400. Craigie,R. and Mizuuchi,K. (1986) Cell, 45, 793-800. Dean,F.B. and Hurwitz,J. (1991) J. Biol. Chem., 266, 5062-5071. Echols,H. (1990) J. Biol. Chem., 265, 14697-14700. Frantz,B. and O'Halloran,T.V. (1990) Biochemistry, 29, 4747-4751. Glasgow,A.C., Hughes,K.T. and Simon,M.I. (1989) In Berg,D.E. and Howe,M.M. (eds), Mobile DNA. American Society for Microbiology, Washington, DC, pp. 637-660. Hatfull,G.F. and Grindley,N.D.F. (1988) In Kucherlapati,R. and Smith,G.R. (eds), Genetic Recombination. American Society for Microbiology, Washington, DC, pp. 357-396. Hatfull,G.F., Noble,S.M. and Grindley,N.D.F. (1987) Cell, 49, 103-110. Heichman,K.A., Moskowitz,I.P.G. and Johnson,R.C. (1991) Genes Dev., 5, 1622-1634. Hughes,R.E., Hatfull,G.F., Rice,P., Steitz,T.A. and Grindley,N.D.F. (1990) Cell, 63, 1331-1338. Johnson,R.C. (1991) Curr. Opin. Genet. Dev., 1, 404-407. Johnson,R.C. and Simon,M.I. (1985) Cell, 41, 781-791. Kahmann,R., Rudt,F., Koch,C. and Mertens,G. (1985) Cell, 41, 771-780. Kahmann,R., Mertens,G., Klippel,A., Brauer,B., Rudt,F. and Koch,C. (1987) UCLA Symp. Mol. Cell. Biol., new series 47, 681-690. Kamp,D., Kahmann,R., Zipser,D., Broker,T.R. and Chow,L.T. (1978) Nature, 271, 577-580. Kanaar,R. and Cozzarelli,N.R. (1992) Curr. Opin. Struct. Biol., 2, 369-379. Kanaar,R., Van de Putte,P. and Cozzarelli,N.R. (1986) Biochim. Biophys. Acta, 866, 170-177. Kanaar,R., Van de Putte,P. and Cozzarelli,N.R. (1988) Proc. Natl. Acad. Sci. USA, 85, 752-756. Kanaar,R., Van de Putte,P. and Cozzarelli,N.R. (1989) Cell, 58, 147-159.
Recombination by enhancer-independent Gin mutants Kanaar,R., Klippel,A., Shekhtman,E., Dungan,J.M., Kahmann,R. and Cozzarelli,N.R. (1990) Cell, 62, 353-366. Klippel,A., Mertens,G., Patschinsky,T. and Kahmann,R. (1988a) EMBO J., 7, 1229-1237. Klippel,A., Cloppenborg,K. and Kahmann,R. (1988b) EMBO J., 7, 3983-3989. Koch,C. and Kahmann,R. (1986) J. Biol. Chem., 261, 15673-15678. Landy,A. (1989) Annu. Rev. Biochem., 58, 913-949. Mertens,G., Fuss,H. and Kahmann,R. (1986) J. Biol. Chem, 261, 15668- 15672. Mertens,G., Klippel,A., Fuss,H., Blocker,H., Frank,R. and Kahmann,R. (1988). EMBO J., 7, 1219-1227. Nash,H.A. (1990) Trends Biochem. Sci., 15, 222-227. Nash,H.A. and Pollock,T.J. (1983) J. Mol. Biol., 170, 19-38. Plasterk,R.H.A. and Van de Putte,P. (1984) Biochim. Biophys. Acta, 782, 111-119. Salvo,J.J. and Grindley,N.D.F. (1988) EMBO J., 7, 3609-3616. Sanderson,M.R., Freemont,P.S., Rice,P.A., Goldman,A., Hatfull,G.F., Grindley,N.D.F. and Steitz,T.A. (1990) Cell, 63, 1323-1329. Stark,W.M., Boocock,M.R. and Sherratt,D.J. (1989a) Trends Genet., 5, 304-309. Stark,W.M., Sherratt,D.J. and Boocock,M.R. (1989b) Cell, 58, 779-790. Surette,M.G. and Chaconas,G. (1992) Cell, 68, 1101-1108. Van de Putte,P., Cramer,S. and Giphart-Gassler,M. (1980) Nature, 286, 218-222. Received on October 9, 1992; revised on November 22, 1992
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