Isolation and characterization of unusual gin mutants.

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Aug 26, 1988 - (Dodd and Egan, 1987) is indicated by broad bars. On the right ..... We are grateful to H.-J.Fritz, K.H.Friedrich and P.Stanssens for providing plasmids .... Mertens,G., Klippel,A., Fuss,H., Blocker,H., Frank,R. and Kahmann,R.
The EMBO Journal vol.7 no.12 pp.3983-3989, 1988

Isolation and characterization of unusual gin

Anke Klippel, Katja Cloppenborg and Regine Kahmann Institut fir Genbiologische Forschung Berlin GmbH, Ihnestrasse 63, D-1000 Berlin 33, FRG Communicated by H.Schaller

Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FISindependent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wildtype situation. Key words: phage Mu/site-specific recombination/random mutagenesis/FIS independence

Introduction In phage Mu a switch in host range is caused by site-specific inversion of the G segment (Kamp et al., 1978; van de Putte et al., 1980). Inversion provides the means to alternate the expression of two sets of genes that are involved in tail fibre biosynthesis (Grundy and Howe, 1984). The recombination sites are 34 bp long and must be arranged as inverted repeats. In direct repeat configuration recombination is barely detectable (Plasterk et al., 1983). DNA inversion is strongly stimulated by a sequence in cis that acts as an enhancer for recombination in a distance- and orientation-independent manner (Kahmann et al., 1985; Schmucker et al., 1986). Recombination reactions performed in vitro have revealed that the DNA substrate must be negatively supercoiled (Mertens et al., 1984; Plasterk et al., 1984). The phagecoded DNA invertase Gin is an essential component of this system. Gin binds to two sites in each IR in a co-operative manner, introduces 2-bp staggered nicks and forms a covalent linkage with DNA at the 5' end of the nick via phosphoserine (Klippel et al., 1988). In the presence of Gin alone DNA inversion occurs with low frequencv both in vitro ©IRL Press Limited, Oxford, England

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and in vivo (Mertens et al., 1986; C.Koch and R.Kahmann, unpublished), suggesting that Gin protein exhibits all catalytic activities. The inversion reaction is > 20-fold stimulated by the Escherichia coli host factor FIS in vitro (Kahmann et al., 1985; Koch and Kahmann, 1986). FIS is a small, heat-stable protein which by its primary sequence is not related to the type II proteins IHF and HU (Johnson et al., 1988; Koch et al., 1988). FIS exists as a homodimer in solution (Koch and Kahmann, 1986) and binds to three sites in the Mu enhancer sequence (C.Koch and R.Kahmann, unpublished). The DNA inversion system of Mu belongs to a family of closely related systems, the other well-studied members being flagellar phase variation in Salmonella typhimurium and host range switching in phage P1 (Zieg and Simon, 1980; Iida et al., 1982). The close relationship is reflected by the interchangeability of the respective DNA invertases, the recombinational enhancers, the requirement for FIS protein and the preference for recombination sites in inverted orientation (see Koch et al., 1987). For the phase variation system it has been demonstrated that the spatial relationship of FIS binding sites is critical for enhancer activity (Johnson et al., 1987) and for the Mu enhancer we have shown that a specific degree of DNA bending induced by FIS is important for enhancer function (C.Koch, F.Rudt and R.Kahmann, in preparation). Such results suggest that the three-dimensional structure of the FIS -enhancer complex may help to assemble the proper synaptic complex via protein-protein interactions between Gin and FIS bound to their respective sites. Alternatively the FIS-enhancer complex could stabilize or select the proper synaptic complex in a negatively supercoiled DNA molecule. Another family of DNA recombinases are the co-integrate resolvases of several transposons. This gene family is distantly related to the DNA invertases. Common features are the need for a negatively supercoiled DNA substrate, the strong preference for intra- rather than intermolecular reactions, the introduction of 2-bp staggered nicks and the covalent linkage to the 5' end of the nick via phosphoserine (Reed and Grindley, 1981; Reed and Moser, 1984). In contrast to the DNA invertases the resolvases have a strong preference for recombination between directly repeated sites. Resolution involves two rather complex res sites but does not require an enhancer type sequence or host factor (see

Sadowski, 1986).

To study the steps in Gin-mediated recombination and in particular to define the role of FIS and the enhancer in this system we have started a genetic analysis by isolating gin mutants that catalyse DNA inversion efficiently in the absence of FIS. This approach became feasible after thefis gene had been cloned and the chromosomal copy had been inactivated by insertion mutagenesis. Such fis mutants are perfectly viable (Johnson et al., 1988; Koch et al., 1988). In this communication we present our initial characterization of several FIS-independent gin mutants.

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A.Klippel, K.Cloppenborg and R.Kahmann

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Fig. 1. Scheme for isolation of gin mutants by chemical mutagenesis. Lesions induced by the mutagen are indicated by small arrows on the strand that carries the mutation. Dots mark amber mutations in bla and cat. The X pL promoter is shown as a black arrowhead; the open arrowhead marks the cat promoter. The open squares indicate the recombinational enhancer sequence; the shaded bar denotes the coding region for the lambda c1857 repressor gene. The gap filling by polymerase is indicated by a broken line. All other elements are indicated in the figure. Details of the procedure are given in Materials and methods and Results.

Results Isolation of FIS-independent gin mutants We have devised a general scheme to isolate mutations in the gin gene by random chemical mutagenesis. The procedure is outlined in Figure 1. The technique is based on the gapped duplex method (Kramer et al., 1984) and uses the pMAC plasmid vectors developed by P.Stanssens et al. (unpublished) to generate random mutations confined to the coding region of gin. A single-stranded template of pLMA5-8gin was incubated with nitrous acid and annealed with a non-mutagenized strand of pMC5-8pL (Figure 1, step 1) to form a gapped duplex molecule exposing only the gin coding region in single stranded form. The gap was closed with Klenow polymerase; in this step only mutations in gin are copied to the outer strand (for details see Materials and methods). After transformation into the repair-deficient strain WK6(XcI+)mutS the population of transformants was grown in the presence of chloramphenicol. The cat gene is intact only in progeny plasmids of the outer strand, the mutagenized inner strand contains an amber mutation in cat. This procedure allows random mutations to be restricted to the gapped region and circumvents the need for recloning mutagenized fragments into non-mutagenized vectors. The last step (Figure 1, step 2) involved transformation of the mutagenized plasmid pool into suitable test strains. Such strains carried the compatible inversion test plasmid pMD31acZ (Klippel et al., 1988) in either fis' or fisgenetic backgrounds. In pMD31acZ (Figure 1) lacZ expression requires inversion. In CSH50[pMD31acZ] up to 70% of the transformants formed white colonies on McConkey lactose plates which indicated a Gin- phenotype. When the same population was transformed into the fis- strain CSH50fis:: Kan[pMD31acZ], 0. 1 % of the transformants formed red colonies at 32°C while the non-mutagenized control plasmid pLMC5-8gin transformed into the same strain produced colonies which stayed white on McConkey lactose plates. Since the time of appearance of the Lac+ phenotype differed between individual transformants from -

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the mutagenized plasmid pool, the gin gene of 10 potential FIS-independent mutants was sequenced. Four mutants had arisen by independent mutations: GinHY106-KG162 (His106 to Tyr, Lysl62 to Gly), GinHY106-NS188, GinTA57KR162 and GinSF75-IV94 (see Figure 2). Since all mutants contained two amino acid alterations the mutations were separated by exchanging suitable restriction fragments of the mutant for wild-type fragments and vice versa (see Materials and methods). Gin mutants generated by this method were tested for recombination in isogenic fisk and fis- strains (Figure 2). The results demonstrate that single amino acid changes in various positions of Gin cause the same phenotype. GinSF75-IV94 is the only mutant where both mutations seemed to contribute to the FIS-independent phenotype, the single mutations both confer FIS independence; however, lacZ expression is delayed by 12 h compared to the double mutant. We have generated four additional mutations (FV104, FV105, FY105 and HT106) in gin by site-directed mutagenesis (see Materials and methods and Figure 2). Of these mutants only GinFV 104 is FIS independent, the other three mutants are gin-. -

In vivo characterization of FIS-independent Gin mutants Plasmids expressing wild-type and mutant Gin proteins were transferred to K12AHIAtrpfis:: Kan. In a second step recombination test plasmids pAK3, pBR25 and pDRl (Figure 5) were introduced. Transformants were grown for at least 36 h at 320C, a temperature that allows only weak expression of gin which is under the control of the X pL promoter (see Figure 1). These conditions were chosen for an evaluation of activity differences of individual mutant Gin proteins; under these conditions equilibrium between the two orientations of the invertible segment is not yet reached. Plasmid DNA was isolated and cleaved with restriction enzymes that permit detection of recombinants; the native DNA was analysed in parallel. pBR25 is an inversion test plasmid that lacks the enhancer, pAK3 is an

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-W0 Fig. 2. Properties of gin mutants and position of changes in the amino acid sequence of Gin. The primary structures of Gin and Tn3 resolvase are aligned on the left according to Grindley et al. (1985). Numbers indicate positions in the Gin sequence. Amino acids that are conserved in the DNA invertase or DNA resolvase family respectively are boxed. Mutant positions are marked by asterisks. The potential helix-turn-helix motive of Gin (Dodd and Egan, 1987) is indicated by broad bars. On the right, individual gin mutants are listed. The property to catalyse DNA inversion in pMD31acZ was analysed in the FIS + strain CSH50 and in the FIS- derivative CSH50fis:: Kan. (+) indicates that inversion activity was reduced. A

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Fig. 3. Comparison of the recombinational activity of several FIS-independent gin mutants in vivo. Plasmid DNA was isolated from a fis- strain carrying both a recombination test plasmid and a plasmid expressing mutant or wild-type Gin protein. M is X DNA cleaved with BstEII. (A) lane 1: pAK3 + pLMC5-8gin; lane 2: pAK3 + pLginHY106; lane 3: pAK3 + pLginKRI62; lane 4: pAK3 + pLginFV104; lane 5: pAK3 + pLginSF75-IV94; lane 6: pBR25 + pLMC5-8gin; lane 7: pBR25 + pLginHY106; lane 8: pBR25 + pLginKRI62; lane 9: pBR25 + pLginFV104; lane 10: pBR25 + pLginSF75-IV94. DNA was cleaved with PstI and analysed on a 2% agarose gel. The band characteristic for inversion is marked by an arrowhead. (B) lane 1: pDRl + pLMC5-8gin; lane 2: pDRl + pLginHY106; lane 3: pDRl + pLginKRI62; lane 4: pDRI + pLginFV104; lane 5: pDRl + pLginSF75-IV94 DNA was cleaved with EcoRI + BamHI, fragments were separated on a 2% agarose gel. The black arrowhead indicates a fragment that arises due to a deletion event, the open arrowhead marks a fragment that disappears when a deletion event takes place. (C) The same DNA as in B was analysed without cleavage. Separation was on a 1% agarose gel. One black arrowhead denotes a plasmid which is the deleted form of the pDR1 monomer. The single open arrowhead marks the monomer starting material of pDR1; two open arrowheads mark dimers of the same plasmid. Two closed arrowheads indicate the position of the dimer in which one deletion event has occurred. Arrows with squares to the right point to higher-order co-integrates.

analogue containing the enhancer. Except for wild-type Gin all mutant Gin proteins catalysed DNA inversion in pAK3 and pBR25 (Figure 3). Relative inversion frequencies in pAK3 and pBR25 were indistinguishable for a given mutant protein. When compared with each other, however, the gin mutants displayed differences in activity. GinKR162 showed the highest activity followed by FV104, HY106 and SF75-IV94. In a fis' background in vivo activities of all mutant proteins measured with pMD31acZ were indistinguishable from wild-type Gin (data not shown). Quite

unexpected was the observation that all mutant proteins could catalyse recombination in pDRl, a plasmid that contains the recombination sites in direct repeat configuration and lacks the enhancer. The deletion frequency varied for different gin mutants, again KR162 showed the highest frequency indicated by the disappearance of a fragment containing the IR sequences (Figure 3B). Essentially the same result was obtained when deletion formation was assayed with plasmid pDR2 carrying the enhancer (data not shown). pDRl plasmids isolated from fis- strains expressing the mutant 3985

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Gin proteins showed an increase in multimeric forms which was most evident for GinKRl62 (Figure 3C). This result implies that in contrast to wild-type Gin the mutant proteins can catalyse intermolecular recombination events as well. The data in Figure 3 suggest, furthermore, that the mutant proteins may catalyse inversions, deletions and intermolecular recombination events with different efficiencies. This aspect will have to be analysed in more detail in timecourse experiments performed in vitro which could eliminate potential artefacts introduced, for example, by the increased replication rate of plasmids which have suffered a deletion.

molecules were used as substrate (Figure 4B). This result prompted us to assay GinKR162 for its ability to recombine plasmid DNA linearized with EcoRI (see Figure 5). Inversion and deletion products were readily detectable with the respective plasmids, and the amount of recombinants was not affected by FIS (Figure 4C). When recombination products of pIR2 were analysed without restricting the DNA, two new fragments were visible (Figure 4D). From their estimated sizes these fragments must represent the products of intermolecular recombination events. These results show that GinKR162 has lost its specificity for sensing the orien-

In vitro properties of GinKR162 Since GinKR162 appeared to be the most active enzyme by all in vivo criteria we have purified it in parallel with the wild-type Gin protein from fis- strains (see Materials and methods). When analysed for inversion in vitro using the

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enhancer containing plasmid pAK3 inversion products were detected with GinKR162, their amount could be stimulated 2- to 3-fold by the addition of FIS (Figure 4A). In pBR25 which lacks the enhancer FIS did contribute to inversion frequency with GinKR162 but to a smaller extent as in pAK3 (Figure 4A). Wild-type Gin showed the expected strong dependence on FIS and enhancer (Figure 4A). Deletion formation assayed with pDR2 was 2-fold inhibited by FIS in reactions containing GinKR162 (Figure 4B). Such an inhibitory effect was not observed when old plasmid preparations which contained a high proportion of nicked -

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tation of the IR sequences as well as the requirement for negatively supercoiled recombination substrate.

Discussion In this study we have used a genetic approach to define amino acid changes in Gin that confer a FIS-independent phenotype. Five such mutations were mapped by sequence analysis, they all lie in different parts of the gin coding region. Four of

these mutations fall into the central region of Gin, a 30 amino acid region between position 75 and 106, while the fifth mutation maps near the carboxyl terminus of Gin (see Figure 2) in a region that has been implicated in DNA binding and is characterized by a helix -tum-helix motive (Plasterk and van de Putte, 1984; Bruist et al., 1987; Dodd and Egan, 1987). The type of amino acid change leading to FIS independence is different for each mutant. Two of the

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mutations, FV104 and HY106, lie in a region of Gin that is highly conserved between the DNA invertases but does not have a corresponding region of similarity in the DNA resolvase family. Position 94 is conserved in both families while position 75 is not even conserved between the DNA invertases (Figure 2). The additional mutations that were introduced by site-directed mutagenesis changed amino acids in Gin into residues found at the corresponding position of resolvase. Only FV104 led to FIS independence. In contrast, the identical exchange Phe to Val at the neighbouring position 105 yielded a recombination-defective protein. A rather conservative change from Phe to Tyr rendered the Gin protein inactive despite the fact that the HY mutation at position 106 conferred FIS independence. A His to Thr change at position 106 which matches resolvase also destroys recombinational activity (Figure 2). In KR 162 only the size of the basic side chain has been altered and this results in FIS independence; GinKG162, on the other hand, behaves like the wild-type protein. These results show that only very specific mutations lead to FIS independence and, furthermore, that at least for some positions in the central domain the affected amino acids and flanking positions are essential for recombinational activity of Gin. From a mechanistic point of view the mutant proteins resemble the site-specific recombinases Cre of phage P1 and FLP of yeast which catalyse inter- and intramolecular reactions on a variety of substrates (Abremski et al., 1983; Meyer-Leon et al., 1984). The activity of the mutant proteins in thefis- background showed quantitative differences, although the types of reactions the mutant proteins could catalyse-inversions, deletions and intermolecular reactions-appeared to be identical. It is conceivable that individual mutations have altered the folding of the protein which could affect stability. We do not consider this a likely explanation because in an isogenic fis + genetic background we have not been able to detect significant differences in inversion activity between individual mutant and wild-type proteins. To us this indicates, furthermore, that the mutations cannot just increase the catalytic activity of Gin. If this were the case we would have expected the mutant proteins to be more active than the wild-type Gin protein in a fis+ background. When inversion was monitored by lacZ expression in pMD31acZ

over time, however, the mutant proteins behaved just like wild-type Gin. If it is unlikely that protein stability is changed or catalytic activity is increased in the mutants what other alternatives do exist to explain their phenotypes? We would like to consider the possibilities that either the binding to DNA, Gin -FIS contacts or Gin -Gin interactions are affected. If DNA binding of the mutant proteins is increased this should again manifest itself as an increase in catalytic activity for which we have no convincing evidence. If the interactions with FIS protein are altered it would make it very difficult to explain that the mutant proteins can still be stimulated by FIS for DNA inversion. Instead this result suggests that the protein domain in Gin which is responsible for these contacts must be intact. Therefore we consider the remaining possibility-effects of the mutations on Gin -Gin contacts as most likely, in particular it does not conflict with any of the results obtained. Gin binds to two sites within each IR in a co-operative manner (Mertens et al., 1988) which presumably involves Gin-Gin contacts. It is presently unclear whether each site is bound by a monomer or dimer of Gin. In any case the step that brings the two IRs together must also involve protein-protein contacts. For wild-type Gin this step may recruit the enhancer-FIS complex to bring the sites together and to align them in the proper conformation for synapsis (Johnson et al., 1987). If the mutations alter the Gin -Gin contacts between the Gin -IR complexes (and individual mutations could do this to varying degrees), the enhancer-FIS complex could become superfluous. On the assumption that the Gin mutants are not affected in their contacts with FIS we can explain why all mutant proteins still respond to FIS and enhancer, although they were selected to catalyse DNA inversion in the absence of enhancer and FIS. Such altered Gin -Gin contacts could result from mutations in different regions of Gin as long as they affect amino acids that are exposed or involved in protein folding. It could also explain how the two mutations in GinSF75-IV94 could both contribute to a FIS-independent phenotype. The new quality of the Gin -Gin contacts could account for the observation that in a supercoiled deletion substrate the formation of deletions by GinKR162 is inhibited by enhancer and FIS. If the FIS -enhancer complex has the dual role of bringing the sites together and imposing

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directionality by aligning the sites in an inversion configuration this will lower the chances of GinKRl62 to establish a synaptic complex without interference by the enhancer and FIS in a deletion substrate. Our data show, furthermore, that the enhancer-FIS complex can impose this directionality (interference) only in a negatively supercoiled DNA substrate, presumably because the negative supercoiling favours the topological specificity for inversion. The mutant Gin proteins are less sensitive to the topologial filter (Gellert and Nash, 1987): due to their altered Gin-Gin contacts they do not need the enhancer and FIS any more for bringing the sites together. This could lead to an increase in flexibility for the formation of productive synaptic complexes not just in inversion but also in deletion substrates and account for the intermolecular recombination events. The proposed dual role of the enhancer - FIS complex in reactions catalysed by wild-type Gin can also be used to explain an observation which so far was difficult to reconcile. The low-frequency formation of deletions with wild-type Gin is strictly enhancer and FIS dependent and furthermore is significantly delayed compared to inversion (R.Kahmann, in preparation). We assume that without FIS and enhancer the sites just would not meet; the enhancer - FIS complex would bring them together but at the same time impose a topology on the complex that is highly unfavourable for deletions. This could explain the delayed appearance of deletion products. Although this discussion must remain speculative, since neither DNA binding studies nor topological analyses have yet been done for the mutant proteins, we are fascinated by the loss of directionality in connection with FIS independence which is achieved by single mutations. Further studies will test our model and determine the functional interactions between Gin-Gin and Gin-FIS in recombination.

Materials and methods Bacterial strains and phages CSH50 (Miller, 1972) is pin- (Kamp and Kahmann, 1981) and was used for the propagation of recombination test plasmids. K12AHIAtrpfis:: Kan was generated by P1 transduction from CSHSOfis:: Kan (Koch et al., 1988) and was kindly provided by C.Koch. WK6(XcI+) and its mutS derivative have been described (Klippel et al., 1988). Helper phage flIR (Dotto and Horiuchi, 1981) was used to prepare single-stranded DNA of plasmids carrying the fl ori. Cloned DNA templates The structure of plasmids used for monitoring recombination in vivo and in vitro is shown in Figure 5. In pAK3 the invertible segment is flanked by 25-bp IR sequences which allow for maximum recombination and the enhancer is present (Mertens et al., 1988). pBR25 is the same construction except for the absence of the enhancer. pIRl and pIR2 both contain the left IR from pBRminiG(-) (Kahmann et al., 1984); a HindIll-BamHI fragment containing a synthetic 34-bp IR was isolated from pAK1 (Mertens et al., 1988) and the protruding ends were filled in and the fragment was cloned in both orientations into the NruI site of pBR322. pIR2 and pDR2 were constructed by cloning the BamHI-EcoRV fragment of pIRI (after filling in the protruding ends) into the EcoRV site of pBR322 in both orientations; in a second step the BamHI-AvaI fragment containing the right IR and the enhancer in wild-type configuration were isolated from pBRminiG(-) (Kahmann et al., 1984) and cloned into the respective pBR322 sites. The plasmids used for mutagenesis of gin, pLMA5-8gin, pLMC5-8gin and pMC5-8pL have been described in detail (Klippel et al., 1988) and are schematically shown in Figure 1. The compatible inversion test plasmid pMD31acZ has been described (Klippel et al., 1988); the schematic structure is again shown in Figure 1. Nucleotide sequence of gin mutants generated by random mutagenesis and separation of mutations 1 refers to the first nucleotide of the gin gene. GinHY106-KG162: C to T at position 316, A to G at positions 484

The nucleotides are numbered:

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and 485; GinHY106-NS188: C to T at position 123 (silent), C to T at position 316, C to T at position 387 (silent), A to G at position 563: GinTA57-KR162: A to G at position 169, A to G at position 485, A to G at position 546 (silent); GinSF75-IV94: C to T at position 224, A to G at position 280. The separation of mutations in gin was as follows: HY106-KG162, a BspMI fragment internal to gin was exchanged for the wild-type fragment, the fragment containing mutation HY106 was cloned into the respective sites of pLMC5-8gin; HYO16-NS188, a Hincdl-HindIll fragment containing NS188, was cloned into the respective sites of pLMC5-8gin. TA57-KR162, an AvaI fragment containing TA57, was cloned into the respective sites of pLMC5-8gin; SF75-IV94, an AseI fragment containing SF75, was cloned into pLMC5-8gin; the corresponding wild-type fragment of pLMC5-8gin was used to replace the fragment in pLgin SF75-IV94.

DNA manipulations Plasmid DNA isolation, ligation, transformation, cleavage of DNA with restriction endonucleases and filling in protruding 5' ends with Klenow polymerase were done according to standard methods (Maniatis et al., 1982). DNA sequence analysis was performed on ssDNA templates by the method of Sanger et al. (1977) using primers described previously (Klippel et al., 1988). The entire gin coding region was sequenced in all mutants generated by chemical or site-directed mutagenesis.

Nitrous acid mutagenesis of gin The majority of deaminations induced by nitrous acid result in C to T and A to G transitions; in addition, several other types of transitions and transversions have been shown to occur (Tessman et al., 1964). The treatment with nitrous acid in vitro was performed according to Myers et al. (1985) with minor modifications: 30 /tg ssDNA, a mixture of pLMA5-8gin and helper phage was incubated in 250 mM sodium acetate, pH 4.4, 1 M sodium nitrite (freshly prepared) at room temperature in a total volume of 100 y1. Aliquots (16 jl) were withdrawn after 2.5, 5, 10, 15, 20 and 25 min. The reactions were stopped by the addition of 3 /g tRNA, an equal volume of 3 M sodium acetate, pH 8.5, 40 Il H20 and 250 A1 ethanol. After 10 min at 0°C the DNA was precipitated. The precipitation step was repeated twice. For the preparation of the gapped duplex molecules - 0.6 jtg mutagenized ssDNA was annealed with - 0.4 jg pMC5-8pL cut with StyI and HindIII. For the gap-filling reaction 75 ng gapped duplex DNA was incubated at room temperature in 100 mM KCI, 15 mM MgCl2, 2 mM DTT, 40 mM Tris-HCI, pH 7.0, 0.125 mM of all four dNTPs, 0.25 mM ATP, 2 U Klenow DNA polymerase and 2 U T4 DNA ligase in a total volume of 40 A1. If mutagens are used which depurinate or depyriminate DNA, the fill-in reactions can be performed under the same conditions except that 20 U AMV reverse transcriptase are used and incubation is at 37°C (A.Klippel, unpublished). With nitrous acid the mutation efficiency measured as occurrence of gin- mutants ranged between 25 and 80% depending on the incubation time.

Site-directed mutagenesis of gin Oligonucleotide-directed mutations in gin were generated as described previously (Klippel et al., 1988) using the gapped duplex approach developed by P.Stanssens et al. (unpublished). The following oligonucleotides were employed. Mutant positions are underlined: FV 104 FV 105 FY105 HT 106

5'AACGTGGAAGACAAAACGCCC3' 5'CCCATAACGTGTACGAAAAAACGCCC3' 5'CCCATAACGTGGTAGAAAAAACG3' 5'CACCCATAACGGTGAAGAAAAAACG3'

Proteins The E. coli host factor FIS was isolated from an overproducing strain (Koch et al., 1988) as described previously (Koch and Kahmann, 1986). Overproduction and purification of wild-type Gin and GinKR162 was from the fis- strain CSHSOfis:: Kan and in principle followed the procedure described by Mertens et al. (1986).

Assays for recombination The in vivo test for assaying the ability of wild-type or mutant Gin proteins to catalyse DNA inversion in plasmid pMD31acZ has been described (Klippel et al., 1988). IacZ expression was monitored on McConkey lactose plates rather than on X-Gal plates after incubation at 320C. To assay recombination in vivo of other plasmids, such substrates were introduced into strains expressing wild-type or mutant Gin protein, DNA was isolated by the cleared lysis method of Bimboim and Doly (1979) and subjected to resriction analysis to reveal fragments characteristic for recombinants. Recombination assays

Isolation and characterization of unusual gin mutants in vitro were performed according to standard conditions (Mertens et al., 1986) using the substrates indicated in each experiment and restricting the DNA to reveal fragments indicative for recombinants.

Acknowledgements We are grateful to H.-J.Fritz, K.H.Friedrich and P.Stanssens for providing plasmids and strains prior to publication. We thank J.Daum, M.Nassal, H.Schaller and L.Willmitzer for the synthesis of oligonucleotides. We acknowledge the efforts of H.Fuss, U.Schafer and S.Kuhl in protein purification and thank Christian Koch, Marlis Dahl, Beate Wittmann, Stefan Maeser, Falko Rudt, Michael Bolker and Gregory Wulczyn for many discussions. This work was supported by the Deutsche Forschungsgemeinschaft (ka 411/2-1).

Sadowski,P. (1986) J. Bacteriol., 165, 341-347. Sanger,F., Nicklen,S. and Coulson,A.R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467. Schmucker,R., Ritthaler,W., Stem,B. and Kamp,D. (1986) J. Gen. Virol., 67, 1123-1133. Tessman,I., Poddar,R.K. and Kumar,S. (1964) J. Mol. Biol., 9, 352-363. van de Putte,P., Cramer,S. and Giphart-Gassler,M. (1980) Nature, 286, 218-22. Zieg,J. and Simon,M. (1980) Proc. Natl. Acad. Sci. USA, 77,4196-4200. Received on July 14, 1988; revised on August 26, 1988

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