two classes tnlo transposase mutants that suppress ... · copyright 0 1996 by the genetics society...

Copyright 0 1996 by the Genetics Society of America

Two Classes of TnlO Transposase Mutants That Suppress Mutations in the TnlU Terminal Inverted Repeat

J. Sakai and N. Kleckner

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 Manuscript received March 11, 1996

Accepted for publication August 2, 1996

ABSTRACT TnlO transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6- 13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named “SEM” for suppression of ends Eutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class I1 mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.

M OBILIZATION of the transposable element TnlO requires only the 402-amino acid TnlO transpc-

sase protein and the 23-bp inverted repeat sequence present at the TnlO termini (BENJAMIN and KLECKNER 1992; CHALMERS and KLECKNER 1994; SIGNON and KLECKNER 1995). The chemical steps of transposition, cleavage and strand transfer at the TnlO termini occur via a nonreplicative mechanism and take place within a protein-DNA complex containing both ends of the transposon and transposase (BENDER and KLECKNER 1986; HANIFORD et al. 1991; BENJAMIN and KLECKNER 1992; SAKAI et al. 1995). The recognition and synapsis by transposase of the two intact TnlO ends into a nu- cleoprotein complex occurs before and is a prerequisite for initiation of the chemical steps (HANIFORD and KLECKNER 1994; SAKAI et al. 1995).

Mutational analysis of the transposon end has shown that the bases most critical for efficient transposition lie in two discrete regions within bp 1 - 13 of the 23-bp inverted repeat (Figure 1A; HUISMAN et al. 1989).

Mutations in the bp 6-13 region confer severe (300- to 2 100,000-fold) decreases in transposition frequency. Analysis of mutant phenotypes, both in vivo and in vitro, has led to the proposal that this region functions as the site of initial recognition of the transposon end by transposase and is required for the formation and stability of the stable complex in which the two transpo-

Corresponding authur: Nancy Kleckner, Department of Molecular and Cellular Biology, Hanard University, 7 Divinity Ave., Cambridge, MA 02138.

Genetics 144: 861-870 (November, 1996)

son ends are synapsed (HUISMAN et al. 1989; HANIFORD and KLECKNER 1994; SAKAI et al. 1995). In particular, the bp 6-13 region has been implicated in close con- tacts with the transposase protein by methylation inter- ference analysis (KLECKNER et al. 1995). Furthermore, mutations in this region confer significant defects in vitro in the level of synaptic complexes formed before cleavage (SAKAI et al. 1995).

In contrast, mutations in bases present in the terminal region of the element (bp 1-3) primarily confer moderate (5 1000-fold) defects in transposition frequency. These mutations appear chiefly to compromise the efficiency of the chemical steps of the reaction (HUISMAN et al. 1989; HANIFORD and KLECKNER 1994; SAKAI et al. 1995); in most cases, the formation of precleavage synaptic complexes is not affected.

We describe here the identification and characterization of IS10 transposase mutations that suppress the transposition defects conferred by mutations in the TnlO termini. Mutations were isolated on the basis of their effects on certain terminus mutations in the bp 6-13 region and then analyzed for their effects on a larger panel of terminus mutations. This analysis has identified two distinct classes of mutations that appear to define two different functional and structural aspects of transposase.

MATERIALS AND METHODS

Media and enzymes: Bacteriological media were prepared as described by MILLER (1972); media for propagation of bact-

862 J. Sakai and N. Kleckner

erophage A were prepared as described by KLECKNER (1979). MacConkey lactose medium was prepared by dissolving 40 g of MacConkey agar base (Difco) and 10 g D-lactose (1%) per liter. When used, ampicillin was added to a concentration of 100 pg/ml, kanamycin sulfate to 50 pg/ml, chloramphenicol to 20 pg/ml, streptomycin sulfate to 150 pg/ml, tetracyline to 12.5 pg/ml. Media components were purchased from Difco; antibiotics were purchased from Sigma. Restriction enzymes, T4 DNA polymerase and T4 DNA Ligase were purchased from New England Biolabs; Sequenase 2.0 was purchased from United States Biochemicals.

Strains and bacteriophages: NK 8032 (Alacpoxlll, recA56,ar- gE,,, Nal', Rif') was used to propagate plasmids; NK5012 (thr-, leu-, lac-, sull, TIR, 15R, 480R) was used to propagate A phages. For matings out, donor strains were NK7378 (recA56, Alacpox. IN, arg-,-,/F'pOX?8) transformed with the appropriate transposon and transposase plasmids used as the donor; NK6641 (recA56, Alacpoxll, a r c , strA) was used as the recipient. NK7378 is described in HUISMAN et al. (1989); NK6641 is described in FOSTER et al. (1981). NK8075 and NK 8027 were used to assay SOS induction. NK 8075 is isogenic to NK8027 but contains a mini-Tn 10 transposon; both strains are described in HANIFORD et al. (1989). NK8034 (argE,,, Alacpowll, recA56, Nal', Rif'/F' lade I-Z fusion Z- Y+ po l l+) was used for all versions of the papillation assay. AJS101, AJS102, and AJSlO6 bearing mini-Tnl0 kanR-'lacZ trasposons with wild type, 8C or 13T terminus mutations were constructed by crossing ANK1038 (i", nin5, hisGD) with pJS179, 180 and 200, respectively (described below); desired products of the cross were identified by screening for Amps Kan' lysogens.

Plasmids: Transposase plasmids: pNK629 is pACYC184 containing the transposase gene driven by the heterologous Ptac promoter; its construction is described in LEE et al. (1987). pJS127 and pJS149 are isogenic to pNK629 but carry the DN35 and DK35 mutations, respectively. pJS138 was constructed by ligating the large EcoRI-StuI fragment from pNK2853 (€€ANI- FORD et al. 1991) to the EcoRI-StuI fragment of pNK629 containing the Ptac-transposase gene. The following are isogenic to pJS138, except they contain a mutation in the GAC codon for amino acid 35, causing an amino acid substitution for the wild-type aspartic acid: pJS 139 (ACC; Thr), pJS140 (CGA, Arg), pJS142 (GCC; Ala), pJS143 (CAC; His), pJS144 (AAA, Lys), pJS146 (CTC; Leu), pJS147 (GAA, Glu), pJS148 (TAT; Tyr) and pJS174 (CCC; Pro). In some cases, other codons coding for the same amino acids were also recovered, for example, both AAT and AAA encoding lysine were recovered.

pNKl688, described in HANIFORD et al. (1989), is a pBR322- based plasmid also containing the transposase gene driven by the Ptacpromoter. pJS175, pJS176, pJS177, pJS263 and pJS264 are isogenic to pNK1688 but contain the mutations VI121, AV127, AT127, LF107 and VI114, respectively, derived by hydroxylamine treatment of pNKl688 and isolated as suppressors of the 8C terminus mutation (below). The following are isogenic to pNKl688 except that they contain the specific transposase mutant derived from the oligonucleotide-di- rected randomization experiments described below: pJS340 (RP106), pJS341 (RH106), pJS342 (RL106), pJS343 (RC106), pJS344 (RA106), pJS345 (RS106), pJS346 (RN106), pJS347 (RT106), pJS360 (LH107), pJS361 (LS107), pJS362 (LI107), pJS363 (LT107), pJS380 (VF114), pJS381 (VC114), pJS382 (VGll4), pJS383 (VR114), pJS384 (VSll4), pJS385 (VAll4), pJS401 (VL121), pJS402 (VR121), pJS403 (VA121), pJS404 (W121), pJS405 (VF121), pJS420 (AD127), pJS421 (AW127), pJS422 (AE127), pJS423 (AC127), pJS424 (AM127). pJS245 was constructed by ligating the XbaI-BamHI Ptac-transposase gene containing fragment to XbaI-BamHI backbone fragment from pACYC184. pJS265 and pJS266 are isogenic to pJS245,

except they bear the transposase genes for AV127 and LF107, respectively.

Transposon plasmids: The basic transposon plasmid used in this study and the mutant derivatives are described in detail in HUISMAN and KLECKNER (1987) and HUISMAN et al. (1989). All are isogenic except for the specific inverted repeat sequence being tested: wt X wt, pNK2456; 1A X lA, pNK3584; 2G X 2G, pNK3585; 3A X 3A, pNK3586; 6T X 6T, pUGM472; 8C X 8C, pUGM568; 9C X 9C, pNK2457; 13T x 13T, pNK2441. Transposon plasmids bearing a specific mutation at only one of the two transposon ends are as follows. 1A X wt, pNK2492; 2G X wt, pNK2493; 3A X wt, pNK2494; 6T X wt, pNKl621; 8C X wt, pNK2496; 9C X wt, pNKl637; 13T x wt, pNK1614.

The plasmids used to cross the mutant transposons onto a A phage were made as follows. pNK157 was made by cloning a BamHI linker into the ClaI site of pNK2242 (SIGNON and KLECKNER 1995); the EcoRI-BamHI backbone from this plasmid was then ligated with an EcoRI-BamHI fragment containing tandem copies of the m B transcription terminator to create pJS158. BgZII fragments containing the kanR-lacZmini- Tnl0 transposon from pNK2456 (wt), pUGM568 (8C), and pNK2441 (13T) were cloned into the BamHI site of pJS158 to create pJS179, 180 and 200, respectively.

Mutagenesis and isolation of SEM mutants: For the isolation of supppressor mutants of the 9C and 13T transposons, pNK629 was mutagenized by hydroxylamine treatment (DAVIS et al. 1980). NK8034 previously transformed with pNK2457 (9C transposon) or pNK2441 (13T transposon) was transformed in 24 or 12 pools, respectively, with mutagenized plasmid DNA. Each pool was plated on MacConkey lactose media containing ampicillin and tetracyline such that there were 50-100 colonies per plate; -24,000 colonies were screened on the 9C transposon, and -12,000 for the 13T transposon. To isolate suppressors of the 8C transposon, pNKl688 was mutagenized by hydroxylamine treatment and transformed into NK8034 lysogenized with AJS102 (8C transposon) in 15 independent pools on MacConkey lactose ampicillin plates; -20,000 colonies were screened. The two systems adjust the papillation level such that transposase mutations conferring small increases in papillation levels are easily discernible.

In both cases, transformants were incubated in the dark at 37"; the plasmid DNA was extracted from those colonies that showed increased levels of papillation relative to wild-type transformants. Potential mutants were rescreened by retrans- forming the extracted DNA into a fresh strain and monitoring the papillation levels on MacConkey lactose plates in which one-half of the plate contained colonies of the potential mutant and the other half contained the appropriate wild-type control transformants. For 17 candidates of 9C and 13T s u p pressors, an EcolU-StuI fragment containing the Ptac promoter and 75% of the transposase gene was subcloned into EcoRI- StuI backbone of pNK2853 and retested for the SEM phenotype. For six candidates, the mutation was precisely localized by sequencing of the promoter and transposase regions by the dideoxy chain termination method of SANCER et al. (1977). Five conferred a strong effect and were the DN35 mutation described in detail below. The sixth mutation was 97T + C; it confers only a twofold increase in transposition regardless of the termini and is located in the putative Shine- Dalgarno untranslated region (RALEIGH and KLECKNER 1986). The phenotype of 18 putative suppressors of the 8C mutation, which conferred at least a fivefold increase were similarly confirmed as above, and the mutations were identified by directly sequencing the promoter and transposase regions.

Assays for transposition frequency: Papillation assay: To determine relative levels of papillation, NK8034 derivatives containing the appropriate mini-Tnl0 transposons were trans-

Suppressors of TnlO End Mutants 863

formed with the test transposases and plated on half plates adjacent to similar transformants containing wild-type transposase. On such a plate, roughly 50 test colonies can be compared to 50 control colonies. Relative transposition levels were determined by directly counting papillae in situations where transposition frequency was sufficiently low (-0-50 papillae per colony). For mini-transposons having different ends mutations, different levels of transposase were required to place the papillation frequency in the appropriate range (see text).

Conjugal “mating out”assay: The mating out assay measures the transposition frequency by measuring the frequency of transposition of a drug resistance-marked transposon into a conjugal plasmid within the donor bacteria. The level of transposition is determined by mating the number of recipient bacteria that have received the drug resistance via the conjugal plasmid after the two cultures have been allowed to mix and mate. This assay was performed essentially as described in HUISMAN et al. (1989). For the mating out assays performed for Tables 1 and 2, the plasmids used to supply transposase were pJS245 (wt), pJS265 (AV127) and pJS266 (LF107) for the class I mutants and pNK629 (wt), pJS127 (DN35), pJS149 (DK35) for the class I1 mutants.

Oligonucleotide “randomization” of individual codons: Specific codons of transposase were mutagenized with degenerate oligonucleotides by the method of KUNKEL (1985). Six oligonucleotides, degenerate for codons 35, 106, 107, 114, 121, or 127, respectively, were synthesized using a Millipore Cyclone DNA synthesizer. Each oligonucleotide contained equal proportions of each base at each position in the targeted codon. These oligonucleotides were used to mutagenize the uracil-substituted single-stranded DNA that was obtained from the wild-type transposase plasmid pJS138 for codon 35 and from pNK1688 for all others. The mutagenesis mixture was transformed directly into NK8034 (AJS106) for the plasmids mutagenized at codon 35 or NK8034 (AJS102) for the plasmids mutagenized at codons 106, 107, 114, 121 or 127. Candidates with SEM phenotypes were picked for all mutagenized codons; candidates that decreased transposition were isolated only for the mutagenized transposases screened on the 8C transposon. Phenotypes were rescreened by papillation on half plates and the mutation identified by sequencing.

S O S induction assay: Mutant transposases isolated from the above randomization procedure were screened for their ability to induce the SOS response dependent on the presence of a wild-type transposon in the chromosome (HANIFORD et al. 1989). In this assay, induction of the SOS response results in the expression of a lacZ gene. Transposase mutants were transformed into both NK8027 and NK8075 and plated on half plates side by side with similar transformants of wild- type transposase. On MacConkey lactose plates, the level of transposase present on wild-type pNK1688 induces the SOS response sufficiently to turn colonies bright red after 48 hr; in the absence of the transposon (NK8027), colonies turn pink over the course of 4 days at 37”.

RESULTS

Isolation of transposase mutations that suppress mutations in the transposon end: Mutations of TnlU transposase were identified that increase the transposition frequency of mini-TnlU transposons bearing a mutation in the bp 6-13 region of the terminal inverted repeat sequences. The corresponding mutations were named SEM for suppressor of ends gutation. Mutant transposases were identified using the lacZ papillation Screen of HUISMAN and KLECKNER (1987) (Figure 1B). In this

A. ; I 0 0

0 0 0 0 0 0 A G A T C C T mutation C T G A T G A A T C C C C T A WT 1 5 10 15 bo

0 . 0 i ;: Seventy of mutant : transposition defect

1 \r I

I I - post- transposase binding synaptic synaptic complex formation steps

B.

f2-3 IS 70 transposase

FIGURE 1.-Transposition system. (A) The TnIO terminus. The mutations in bold are the terminus mutations for which suppressor mutations were isolated. Decreases in transposition frequency conferred by specific terminus mutations are from HUISMAN et al. (1989). Each filled oval corresponds to a lOfold decrease in transposition frequency. Bp 1-3 and 6- 13 have been proposed to be discrete functional domains. (B) Papillation transposition assay system. Transposase was provided on a plasmid that was mutagenized by hydroxylamine treatment to generate suppressor candidates. The appropriate transposon, either wild type or bearing a single terminus mutation in both transposon ends, was present on a separate replicon (see MATERIALS AND METHODS for details). In addition, transposons bearing a single terminus mutation in only one of the two transposon ends were used for the analysis in Table 2. lTTT symbolizes four tandem repeats of the rrnB operon strong transcriptional terminators, which are present in the starting configuration to prevent externally promoted transcription of the lac2 gene.

assay, the mini TnlOlacZ element does not express 0- galactosidase in the starting configuration due to the absence of transcriptional and translational start sig- nals. Transposition of the element into an actively tran- scribed region can activate ZacZexpression. In a suitable strain background, these transposition events can be detected in individual colonies growing on MacConkey lactose agar plates as red, Lac’ papillae. The number and time of the appearance of the papillae has been directly correlated to the transposition frequency (HUISMAN and KLECKNER 1987).

To screen for suppressor transposases, a plasmid carrying the TnlO transposase gene fused to a heterologous promoter was mutagenized in vitro with hydroxylamine and introduced by transformation into a strain carrying the mini TnlOlacZ fusion transposon with appropriate mutant termini. Potential mutants were identified as colonies that had a higher than average number of papillae (MATERIALS AND METHODS). Candidates were confirmed as mutants by extraction of the plasmid DNA and retransformation into a fresh strain background to verify the mutant phenotype.


Closs I: Transposase mutants were sought using a mini-TnIO element bearing the 8C mutation at both ends. In combination with wild-type transposase, this mutation confers a 300-fold decrease in transposition frequency (HCISM~\N P/ nl. 1989). Approximately 20,000 colonies were screened and 18 putative suppressors were identified that increased the transposition frequency of the mutant transposon five- to 10-fold. DNA sequence analysis of the transposase genes in these plasmids revealed that the 18 mutants represent five different mutations that map to four codons. The mutations isolated were as follows: LF107, VIll4, VI121, AV127 and AT1 27.

Clnss 11: Transposase mutants were also sought using mini-TnIO elements bearing the mutations 9C or 13T at the two transposon ends. In combination with wild- type transposase, these mutations confer decreases in transposition frequency of 5000- and 2000-fold, respectively. From the 24,000 colonies screened on 9C and the 12,000 colonies screened for suppressors o f 13T, four independent candidates were identified that increased the transposition frequency on the mutant transposons 10-fold. All four mutations changed the aspartic acid at position 35 to asparagine (DN35).

Overall suppression patterns for the class I and class I1 mutations on mutant transposon termini: The five different class I SEM mutants (LFl07, VI114, VI121, AT127 and AV127), the original class I1 SEM mutations (DNS5) and a second allele at the same position that confers a stronger suppression (DK35; see below) were screened by the papillation assay for their effect on a panel of mini Tn IOHrrZ elements containing different terminus mutations (Figure 2). In addition, for two class I mutations, AV127 and LFlO7, and two class I1 mutations, DN35 and DK3.5, transposition frequencies for the panel of mutant transposons have been determined quantitatively using a mating out assay (Table 1 and Figure 3 ) .

l h froo rlnssPs qf SEM / r m s j ~ o s o x s hnrw rommon Jk- 17ir~s: All of the SEM mutations appear to sharc three important characteristics displayed visually in Figure 2. First, the SEM mutations have little to no effect on the transposition frequency of the wild-tyx transposon. Second, each SEM mutation suppresses the defect(s) of terminus mutation(s) other than that upon which it was isolated. Third, suppression effects tend to be larger (five- to 10-fold) for transposons bearing mutations in the bp 6-13 region and smaller (usually at least threefold) for transposons bearing mutations in bp 1-3. In some cases, particularly for the AVl27 transposase, the transposition frequency on the bp 1-3 mutations is even decreased relative to wild type.

T h /700 r l m s ~ s qf.WM mulolions Pxhibil di [ f im/ supjms- sion pnl/Pms: Both papillation and the quantitative mating out assay show that the patterns of suppression of the terminus mutations exhibited by the SEM mutants are generally similar to each other if the SEM mutations

?.

Relative transposition frequencies ~~~ ~ ~~~

Transposase Transposon Ends WT 1A 2G 3A 6T 8C 9C 13T

WT

LF107

VI1 14

VI121 AT1 27

AV127

Class I

Class II DN35 DK35

0 0 0 0 0 0 0 0

0 0 0 . 0 a o . O 0 0 8.80 0 0 0 6.60 0 0 @ . @ O 0 0 0 0 0 - 8 0

00.2-0.5~ OWt 0 3 @5-1OX -20-100X

FIGURI.: P."S~rppression hy the SEM transposases of the transposition defects conferred hy the terminus mutations. Transposition frequencies were assayed qualitatively hv the papillation assay for painvise comhinations of mutant transpo- Sase and mutant ends and compared to the papillation le~els observed for wild-type transposase on the same mutant ends. In each case, the comparison was done on -20-.50 colonies of each type spread on a semicircle of a single MacConkev lactose plate. Transposition frequency has been previously determined to correspond to the level of papillation observed (Hcrsal,\N c/ ol. 1989). The relative difference in papillation frequencies is indicated by the colored circles. Transposition frequencies as estimated hy these tests correspond closely to those defined more accurately by quantitative analysis (com- pare with Tahle 1).

are from the same class, but the patterns of the two classes differ significantly from each other (Figure 2 and Table 1). The suppression effects of the class I mutations are strongest for the 8C mutation upon which the mutants were isolated and moderate for two immediately acijacent mutations 6T and 9C; at other positions, suppression is generally either nonexistent or negative ( i . P . , the mutant transposase is less effective than wild type). In contrast, the class I1 mutations exhibit strong suppression effects on 6T, 9C and 13T, while more moderate suppression effect$ are observed for mutations within bp 1-3 and for mutation 8C.

For wild-type and selected mutant transposases, the relative transposition frequencies conferred by different terminus mutations can be compared graphically (Fig- ure 3 ) . For the class I mutations, the hierarchy of different terminus mutants with respect to transposition frequency is substantially rearranged from that observed with wild-type transposase. This effect reflects the fact that these mutations selectively suppress certain terminus mutations and not others. For the class I1 SEM mutations, in contrast, terminus mutations exhibit essentially the wild-type hierarchv, but the range of transposition frequencies represented is less. This effect reflects the fact that class I1 mutations confer an increase in transpo-


TABLE 1

Transposition frequencies of class I and class I1 SEM mutants on mini-TnZO transposons

Transposon ends

Transposase WT 1A 2G 3A 6T 8C 9 c 13T

Class I WT =1.0 0.001 0.03 0.004 0.0005 0.006 0.00003 0.0004 AV127 1 .0 0.0002 0.008 0.002 0.003 0.24 0.0001 0.0003 LF107 1.3 0.0004 0.04 0.01 0.003 0.08 0.0002 0.0006

Class I1 WT =1.0 0.0008 0.026 0.0027 0.0001 0.003 ND" 0.00013

DN35 2.1 0.0026 0.086 0.0072 0.009 0.01 ND 0.0013 DK35 0.8 0.0047 ND ND 0.0035 0.028 ND 0.0075

~ ~ ~

Absolute transposition frequencies were measured as Kan' exconjugants/ml mating mixture (see MATERIALS AND METHODS) and then normalized to the value obtained for the wild-type transposon/wild-type transposase combination in the same experi- ment. When these experiments are performed properly, absolute exconjugant frequencies vary by < ?20% both within the cultures analyzed on a given day and even from day to day. Class I and class I1 mutations were analyzed in two separate sets of experiments. Eight to 35 cultures of each transposase/transposon combination were analyzed in each case; results are averaged from a total of nine independent experiments for class I and a total of five independent experiments for class 11. The average values obtained in the two sets for the wild-type transposon/transposase combination were 3.7 and 3.5 X lo" Kan' exconjugants/ ml respectively. In this analysis, any difference in relative transposition frequency of 220% or more is significant.

~~

a ND, not determined. '

sition of all mutant elements tested, but have greater effects on elements carrying terminus mutations in bp 6-13 than on those carrylng mutations in bp 1-3.

Class I and class I1 SEM mutations behave differently with respect to suppression effect on single end mutant transposons as compared to double end mutant transposons: In HUISMAN et al. (1989), an analysis was performed using wild-type transposase that compared the extent of the decrease in transposition frequency conferred by a given terminus mutation when present at either one or both transposon ends. This analysis re-

Class I 6T

WT 2G 8C3A 1A 13T 9C

wT " AV127 4-I-w-b LF107 -U-l-c&w-

Class I I WT fl DN35 -- DK35 - -

I . .....- I , "'....I . . ' . .".I ' . * " ' " I ' ""." I ' ""-7 .I 1 10 100 1000 10000 100000

relative transposition frequency

FIGURE 3.-Hierarchy of the terminus mutations for wild- type and SEM transposases according to transposition frequency. For wild-type and selected SEM transposases, transposition frequencies for mini-TnIO elements bearing different terminus mutations are presented graphically with each particular terminus mutation indicated by a specific symbol as defined in the top line. Terminus mutations within bp 6-13 are denoted by filled symbols; terminus mutations within bpl-3 are denoted by open symbols. Data are from Table 1.

vealed that the defects conferred by the mutations in bp 6- 13 were distinct from those in bp 1-3. In particular, the decrease in transposition frequency of the doubly mutant bp 6- 13 transposons was roughly the square of the decrease observed for the singly mutant transposons; from this correspondence it was inferred that each mutant end contributed independently to the defect in the doubly mutant element. For each mutation in bp 1-3, in contrast, the square of the singly mutant transposon defects was much less than the observed defect for the corresponding double mutant transposons. Since this observation suggested that the presence of a wild-type end could compensate in part for the defect conferred by any bp 1-3 mutation at the other transposon end, it could also be inferred that the defect in bp 1-3 mutations occurred subsequent to an interaction between the transposon ends. These predictions have been confirmed by subsequent analysis: bp 6-13 mutations affect formation of the transposase synaptic complex, while bp 1-3 mutations primarily affect chemical steps that occur subsequent to synaptic complex formation (KLECKNER et al. 1995; Introduction).

We have used a similar rationale to determine whether the suppression effects exerted by SEM mutations occur after an effective end/end interaction or before (or during) such an interaction. We analyzed the suppression effects of selected class I and class I1 SEM transposases on singly and doubly mutant mini- TnlO elements as described in Table 2.

The two classes of SEM mutations give different results in this analysis. The class I SEM mutations give no evidence of end-autonomous suppression. The magni- tude of the suppression effect conferred by the class I SEM transposases is the same or similar for both singly and doubly mutant elements. We infer that class 1 muta-


TABLE 2

Relative suppression values for SEM transposases on mini-TnlO elements carrying a mutation at one end (SE) or both ends (DE)

Transposon

Transposase

WT Class I

AV127 LF107

Class I1 DN35 DK35

2G 3A

WT SE DE SE DE

=1.0 =1.0 =1.0 =1.0 = 1.0

1.03 0.5 0.3 0.5 0.5 1.3 1.4 1.4 1.2 0.4

2.1 2.0 3.3 2.9 2.6 0.8

6T

SE DE

8C 13T

SE DE SE DE

=1.0 =1.0

13.3 6.8 8.4 6.7

2.0 9.0 4.0 35.0

=1.0 =1.0 =1.0 =1.0

4.8 40.9 1.2 0.8 7.6 14.2 1.4 1.9

3.6 10.0 8.3 57.7

The abilities of each particular mutant transposase to suppress different terminus defects, when present at either one (SE; single end) or both ends (DE; double end) of a mini-Tnl0 element, are shown. For each combination of transposase and transposons, a “relative suppression value” is given. This value describes the extent of the ability of a particular mutant transposase to suppress a particular terminus mutation: the transposition level observed with a particular mutant transposase on a particular mini-Tnl0 element is divided by the transposition level observed with wild-type transposase on that same mini-Tn10 element. The greater the extent of suppression by the mutant transposase, the higher this ratio will be. For DE mini-transposons, the two component values are taken directly from Table 1 (the ratio of the relative transposition frequencies will be the same as the ratio of the absolute transposition frequencies). For example, for mini-transposon 2G DE, relative transposition frequencies with mutant and wild-type transposase are 0.086 and 0.026, and the corresponding relative suppression value is 3.3. In some cases, values in Table 2 differ slightly from those predicted from Table 1 due to rounding off of numbers in Table 1. For SE mini- transposons, component values were taken from an exactly analogous set of data obtained in an exactly analogous way with the appropriate transposon substrates. When the relative suppression values observed for SE and DE mini-transposons (and a given mutant transposase) differ by more than twofold, both values are highlighted in bold. In such cases, it is inferred that the suppression effect acts separately on the two termini of the element and thus that the suppression effect occurs before or during the establishment of a stable interaction between the two ends. For mutant transposases in which significant suppression is observed but the relative suppression values differ by twofold or less, it is inferred that the suppression effect acts on the two ends coordinately and thus that the suppression effect occurs after establishment of a stable interaction between the two ends.

tions likely exert their suppression effects after end/ end interaction (e.g. , synaptic complex formation). There is one apparent exception to this generalization. For the combination of the SEM transposase AV127 and the 8C mutation, the transposition frequency of the 8C X 8C doubly mutant element is increased 40- fold while that of the singly mutant 8C X wt transposon is increased fourfold. In this case, however, the ex- pected result could be masked by a threshold effect: a 40-fold increase in the singly mutant case could not have been observed because this element exhibits only a fourfold deficit in transposition.

The class I1 mutations seem to suppress mutations in the two ends independently. On the transposon con- structs bearing mutations in the bp 6-13 region, in particular 6T and 13T, the increase observed on the transposon with the mutation in both ends is close to the square of that observed on the singly mutated transposon (Table 2). This effect could, however, be specific to mutations in bp 6-13, i.e., those most strongly affected by the class I1 SEM mutations: for the transposons bearing mutations 2G or 3A the class I1 DN35 transposase confers two- to threefold suppression effects on both singly and doubly mutant elements.

Randomization of codons identified by class I and class I1 SEM mutants: For each of the codons identified as the site of an SEM mutation, the spectrum of amino

acid residues that could confer an SEM phenotype was further investigated by saturation mutagenesis. Each codon was mutagenized specifically using an oligonucleotide with equal frequencies of the four bases at each of the three positions. Each individual pool of mutagenized transposases was screened by the papillation assay to identify those exhibiting altered frequencies of transposition with an appropriate mutant mini TnlUlacZ element, 8C for the class I codons (positions 107, 114, 121 and 127) and the 13T transposon for the class I1 codon (position 35).

At class I codons, SEM alleles contain hydrophobic residues: The four class I SEM mutations map about seven codons apart and all specify hydrophobic amino acids. Further, the SEM mutations recovered usually substitute a hydrophobic amino acid for the original amino acid. These observations suggest that this region defines a short region of a-helical coiled-coil within transposase.

This view is supported by the randomization analysis. For each class I codon, 300 mutagenized transformants were examined; a few percent exhibited increased transposition of the test element. All such candidates were rescreened, and those that retained the SEM phenotype were analyzed by DNA sequencing. For each codon, all the SEM alleles contain a hydrophobic amino acid that is similar in size o r slightly larger than the

Suppressors of TnlO End Mutants

TABLE 3

S u m m a r y of phenotypes conferred by amino acid substitutions at specific SEM and nearby codons

Phenotype

Codon SEM on 8C SOS-Tnsp- SOS’Tnsp-

L107 Ile, Phe His, Ser, Thr None V114 Ile Ser, Gly, Arg None

A127 Val, Thr, Cys Met, Asp, Glu, Trp None R106 None Pro His, Asn, Glu, Leu, Cys, Ser, Ala, Thr

v121 Ile, Leu Tyr, Ala, Arg None

867

amino acid present at that position in the wild-type protein (CREIGHTON 1984; Table 3). At codon 107, leucine could be replaced by isolecine, or in the original hydroxylamine-derived allele by phenylalanine; at codon 114, valine could be replaced by isoleucine; at codon 121, valine could be replaced by isoleucine and leucine; at codon 127, alanine could be replaced by valine, cysteine or threonine.

A t class 11 codon 35, the SEM phenotype involves a charge interaction: For the class I1 codon 35, -500 mutagenized transformants were examined; more than half exhibited increased transposition on the test element. Twenty-nine candidates representing the entire range of suppression levels were retested and sequenced. These comprised 11 different alleles that confer increases in transposition frequency of the 13T transposon that vary over a wide range, from 2.5- to 80-fold (Figure 4). At this codon, the most important variable appears to be the charge of the amino acid. The amino acid present in wild-type transposase at this position is the negatively charged residue aspartic acid. In contrast, the three SEM alleles that confer the strongest suppression effect, 60- to 80-fold, encode the three positively charged amino acids histidine, lysine and argi- nine. Correspondingly, the SEM alleles that confer weak effects either encode the other negatively charged amino acid, glutamic acid, or a large hydrophobic residue, leucine, tyrosine or phenlalanine. Alleles that confer moderate effects, five- to 15-fold, include asparagine, threonine, proline and alanine.

Codons that yield class I SEM mutations are probably distinct from codons that yield SOS+Tnsp- or ATS mutations: The class I SEM mutations are embedded in Patch I [amino acids (aa) 97-1671, a region of transposase containing mutations that confer other types of phenotypes: “SOS’Tnsp”’ mutations that permit excision of the transposon but are defective for insertion into a target site (IIANIFORD et al. 1989), and an “ATS” mutation that relaxes the specificty of TnlO insertions for particular target DNA sites (BENDER and KLECKNER 1992). One SEM mutation, LF107, is immediately adjacent to the SOS+Tnsp- mutation RQ106.

The three different phenotypes are, per se, distinct. ATS mutations do not confer an SOS+Tnsp- phenotype, or vice versa. Furthermore, neither the ATS muta-

tion CY134 nor any of the 10 SOS+Tnsp- alleles examined confer an SEM phenotype for transposons bearing terminus mutations 8C or 13T. Nor do SEM mutations confer transposition defects as do all SOS+Tnsp- mutations.

In some cases, the ATS and SOS’Tnsp- phenotypes are related. An ATS mutation at one position can suppress an SOSfTnsp- mutation at another position, and more strikingly, the two phenotypes can arise as the result of two different substitutions at the same amino acid position ( JUNOP et al. 1994). As part of the current study, we examined the relationship between SOS+Tnsp- and SEM phenotypes by detailed analysis of mutations at codons 106 and 107.

x 1001 1 u S 13T transposon a 3 80- 0- 2

60- S 0 “ .- + v) 40-

S 20-

0 n

E wt transDoson I

Asp Leu Tyr Glu PheThr Asfi Ala Pro Arg HisLys

(wt) Amino acid at codon 35 FIGURE 4.-Relative transposition frequencies of class I1

SEM transposases recovered from the randomization analysis. Codon 35 was mutagenized by saturation mutagenesis and the amino acid substitutions that confer an SEM phenotype were identified by the papillation assay and confirmed by retesting and sequencing. The relative transposition frequencies for those transposases were measured for both wild-type and the 13T mutant transposon by the mating out assay. Wild- type transposase on wild-type ends gave a value of 7.4 X l o 5 Kan‘ exconjugants per ml of mating mixture; wild-type transposase on 13T ends gave 1.6 X 10‘ exconjugants per ml of mating mixture. * denotes the original hydroxylamine-derived mutant.


At the four class I SEM codons, a mutation appears to confer either an SEM phenotype or a general transposition defect, but does not confer an SOS+Tnsp- phenotype (Table 3). The pools of transposase genes mutagenized specifically at the four class I codons were further screened for the presence of mutations that confer transposition defects; for convenience, mutants were identified in the same screen used to search for additional SEM alleles using the 8C mutant mini TnlU lac2 element. From each pool, 12 transposition-defective transposase mutants were identified and assayed in combination with either a wild-type element or the 8C mutant element for transposition frequency by lac2 papillation and for SOS induction in a previously described lac2 turn on assay (HANIFORD et al. 1989). These mutant transposases generally conferred similar defects on both the wild-type and 8C elements, and none of the transposition defective mutants exhibited a discernible level of SOS induction.

Analogous analysis was carried out using a pool of transposase plasmids mutagenized specifically at codon 106. Among 300 transformants, none exhibited an SEM phenotype; among 12 transposition defective mutants, 11 exhibited an SOS’Tnsp- phenotype, and one exhibited an SOS-Tnsp- phenotype.

These observations suggest that despite their close physical proximity, residues 106 and 107 are involved in two different aspects of protein function: SEM and SOS’Tnsp- phenotypes are likely to be distinct. Al- though alleles of the class I SEM codons do not give rise to the SOS+Tnsp- phenotype, not all SOS+Tnsp- codons are functionally equivalent ( JUNOP et al. 1994; KLECKNER et al. 1995). Thus, it remains possible that substitutions at another SOS+Tnsp- codon more dis- tant from the SEM mutations than codon 106 may be able to confer the SEM phenotype. Nonetheless, residues 106 and 107 appear to be involved in two different processes despite their close physical proximity.

DISCUSSION

SEM mutations suppress the defects of TnlO terminus mutations via qualitative changes in IS10 transposase protein: We have identified mutations in IS10 transposase that partially suppress defects conferred by mutations in the termini of Tn 10.

Each of these “SEM” transposase mutations suppresses the defects conferred by several different terminus mutations; moreover, none of the mutations has any dramatic effect on transposition of wild-type transposon. Thus none of the SEM mutations confers a simple alteration of transposase/end recognition specificity.

Furthermore, none of the mutations exerts its effects by increasing the level of transposase protein. A mutation in the transposase Shine-Dalgarno region, which should affect only the quantity and not the quality of

transposase, coordinately increases the frequency of wild type and all mutant end transposons similarly, about twofold (data not shown).

Finally, none of the SEM mutations has much affect on the transposition of a Tn10 element with wild-type ends. Furthermore, none of the SEM mutations affects either preferential cis action of transposase or the in- verse relationship between transposition frequency on transposon length (MORISATO et al. 1983; data not shown). Thus, these suppressor mutations are compen- sating for a defect created by the terminus mutations rather than affecting an aspect that limits the frequency of wild-type transposition reaction.

Two distinct types of SEM mutants have been identified, as follows:

SEM class I: Class I transposase mutations appear to define a single small structural and functional domain of transposase. All of these mutations exhibit the same specific suppression pattern with respect to the assayed terminus mutations, affecting mutation 8C strongly and mutations 6T and 9C to a lesser degree, and all of these mutations occur at one of four codons that map near one another, residues 107, 114, 121 and 127. In addition, although this series of codons is contained within a larger region that gives rise to mutations affecting donor DNA cleavage, target recognition and strand transfer (Patch I; HANIFORD et al. 1989; BENDER and KIECKNER 1992;JUNoP et al. 1994; BOLLAND and KLECK-

NER 1995; KLECKNER et al. 1995), the SEM phenotype appears to be distinct. None of the SOS+Tnsp- or ATS mutations located in Patch I confer an SEM phenotype and, conversely, randomization analysis of SEM codons, though not exhaustive, did not reveal substitutions that conferred an SOS’Tnsp- phenotype.

It seems likely that the domain defined by class I mutations is involved in a hydrophobic interaction that is essential for transposition activity but can confer an SEM phenotype if slightly modified. The four codons at which these mutations occur all encode hydrophobic amino acids in the wild-type protein and all of the SEM alleles substitute a similar or slightly larger hydrophobic amino acid, while substitution of other types of amino acids confers transposition defect.

The relevant hydrophobic interaction is probably required after the two transposon ends have interacted with one another but before the first chemical step of transposition, ie., transposase binding and/or synaptic complex formation. Class I SEM mutations seem to suppress the defects of singly and doubly mutant termini similarly, which suggests an effect subsequent to end- end interaction. And mutations at class I SEM codons that confer transposition defects all block SOS induction, i.e., transposon excision, as well as full transposition, which implies a defect before cleavage. Similarly, two other mutations that lie among the class I SEM mutations, but at different codons, also appear to affect


the reaction before cleavage (JLECKNER et al. 1995; BOL LAND and KLECKNER 1996).

It is further possible that the cluster of class I SEM mutations in fact defines a relatively self-contained functional domain of transposase protein. Among the many mutations that affect later steps of the reaction, none map within residues 107-121, even though many map just to either side of the class I SEM codons as well as at other positions in transposase.

The four class I SEM codons are located roughly seven amino acids apart and reside in a potentially alpha-helical region (data not shown) and thus could be part of an alpha helical coiled coil. The predicted alpha helix would not be amphipathic, however, nor would it exhibit the canonical features of a leucine zip per (Hu et al. 1993). It would also be unrelated to a putative leucine zipper motif of unknown functional significance identified near the carboxy-terminus of IS50 transposase (WEINREICH et al. 1994).

SEM class IZ: The class I1 SEM mutations comprise alleles of a single codon near the amino terminus of the protein, residue 35. SEM alleles at this position increase the transposition frequencies of all mutant transposons tested and the hierarachy of relative transposition frequencies of the various mutant elements is not affected. Thus, the effects of class I1 SEM mutations are distinct from, and much more general than, the effects of class I SEM mutations.

Class I1 SEM mutations might also exert their suppression effects at early stages of the transposition reaction. Despite their relatively broad spectrum of effects, these mutations still have a greater effect on terminus mutations in bp 6-13, the primary transposase interaction region, than on terminus mutations in bp 1-3, which are likely important for chemical steps (most importantly, strand transfer.) Furthermore, class I1 mutations, in contrast to class I mutations, may exert their suppressive effects independently at the two transposon termini. Thus, these mutations might act by stabilizing transposase-terminus interactions during the earliest stages of synaptic complex formation, before interaction between the ends (and before the step affected by class I mutations).

The class I1 SEM phenotype appears to result from a change in a charge interaction. In wild-type transposase, residue 35 is negatively charged and the strongest SEM phenotypes result from substitution of each of three residues that can be positively charged. A change in a charge interaction appears to be the basis for several other increase-of-function mutations (e.g. , Trp superrepressor, %IC and YANOFSKY 1988), but intermediate SEM phenotypes result from substitution of diverse other residues that do not seem to share any chemical characteristics. Apparently, simple disruption of an in- hibitory interaction confers some benefit while substitution of a favorable interaction confers maximal benefit.

The site of class I1 mutations, amino acid 35, lies

within an amino-terminal structural domain of transposase defined by proteolysis studies. This domain is not absolutely required for formation and activity of the synaptic complex: proteolytic fragments lacking this domain can still form functional synaptic complexes in vitro; such complexes form at somewhat reduced levels, however, and exhibit somewhat reduced specific activity, which suggests that the amino-terminal domain is important for fully normal transposase function (KWON et al. 1995). In accord with the relative unimportance of this region for the wild-type reaction, none of the varied substitutions at residue 35 analyzed here alter the transposition frequency of a wild-type element by more than twofold.

Summary: Class I and class I1 SEM mutations compensate for defects conferred by transposon end mutations but have little effect on transposition by a wild-type Tn 10 element. The two classes of suppressor mutations affect different regions of the protein and act in two distinct ways, both of which appear to involve steps before the first chemical event of transposition. Class I mutations occur in residues 107-121, likely affect a hydrophobic interaction between two alpha-helical regions, and appear to define a small functional and structural domain of the protein that is required after the two transposon ends interact but before the first chemical step. Class I1 mutations occur specifically at residue 35, within the helpful but nonessential amino-terminal domain of transposase, appear to affect the reaction by changing a charge interaction, and seem to affect an extremely early step in the reaction, one that precedes establishment of an effective interaction between the two transposon ends.

We thank members of the KLECKNER laboratory both past and present, as well as members of the HANIFORD lab for their interest in this work and comments on the manuscript. In particular, we thankJUDITH BENDER, LAURENCE SIGNON, CHAITANYA JAIN, ANTHONY

SCHWACHA, DAVID HANIFORD and SEAN BURGESS for interesting discus- sions during the course of this work and for generously sharing materials on occasion. This research was supported by a grant to N.K. from the National Institutes of Health (GM-25326).

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