Proc. Natl. Acad. Sci. USA Vol. 90, pp. 2925-2929, April 1993 Biochemistry (Tral) of IncPa RP4 catalyzes a site-specific cleaving-joining reaction of single-stranded DNA (/active-site /sequence-specific recognition of single-stranded DNA/termination of transfer DNA replication) WERNER PANSEGRAUt, WERNER SCHRODERt, AND ERICH LANKAt§ tMax-Planck-Institut fur Molekulare Genetik, Abteilung Schuster, Ihnestrasse 73, D-1000 Berlin 33, Federal Republic of Germany; and tFreie Universitat Berlin, Institut fur Biochemie, Fabeckstrasse 36a, D-1000 Berlin 33, Federal Republic of Germany Communicated by F. William Studier, December 14, 1992

ABSTRACT Conjugative DNA transfer of the self- (TraI) to nic. The role of TraH is to stabilize the initial transmissible broad-host-range plasmid RP4 is initiated by complex of form I oriT DNA, TraJ, and TraI by specific strand- and site-specific cleavage at the site (nic) of the -protein interactions (2). transfer origin (oriT). Cleavage results in covalent attachment We report on the mechanism of site- and strand-specific of the plasmid-encoded relaxase (TraI) to the 5'-terminal cleavage at oriT as part of a study of components involved in 2'-deoxycytidine residue at nic. We demonstrate that Tyr22 is the initiation and termination processes of RP4-mediated the center of the catalytic site of TraI, mediating cleavage via DNA transfer. The specific interaction of RP4 relaxase with formation of a phosphodiester between the DNA 5' phosphoryl defined oligodeoxyribonucleotide substrates served to de- and the aromatic hydroxyl group. The specificity of cleavage scribe (i) the cleavage and rejoining reaction, (ii) the nucle- seen with form I oriT DNA was verified with short oligodeoxy- otide specificity within the nick region, and (iii) the nature of ribonucleotides embracing the nick region. The reaction re- the covalent bond between the DNA and the relaxase. To test quires TraI and Mg2+ but is independent of the relaxosome the hypothesis that termination of transfer DNA replication component TraJ. Cleavage produces one oligonucleotide frag- occurs by site-specific recombination between single- ment with a free 3' hydroxyl, the other part forms a covalent stranded oriT sites, we employed purified TraI of plasmid Tral-oligonucleotide adduct. Like nicking of form I oniT DNA, RP4 in an in vitro assay detecting strand exchange at nic. Tral-catalyzed oligonucleotide cleavage reaches an equilibrium Consideration of analogous DNA sequences in a variety of when about 30% of the input Tral exists as a covalent protein- other DNA transfer systems, including the agrobacterial Ti DNA complex. In the presence of two differently sized oligo- , leads us to suggest that the mechanism described , defined hybrid oligonucleotides are produced, here is a widespread mode ofgene transmission between both demonstrating that TraI catalyzes recombination of two single closely and remotely related organisms. strands at nic. This rmding shows that TraI possesses cleaving- joining activity resembling that of a type I . Reactions are dependent on the sequence of the 3'-terminal 6 MATERIALS AND METHODS nucleotides adjacent to nic. Only certain base changes in a few and DNA. TraI and TraIA3 were purified as positions are tolerated, whereas the sequence of the 5' terminal were labeled either nucleotides apparently is irrelevant for recognition by Tral. described (2). Oligodeoxyribonucleotides The reactions described here further support the hypothesis at their 3' ends by using [a-32P]ddATP (3000 Ci/mmol; 1 Ci that DNA transfer via conjugation involves a roiling circle-like = 37 GBq) and terminal deoxynucleotidyltransferase (Am- mechanism which generates the immigrant single strand while ersham) or at their 5'-ends by using [y-32P]ATP (3000 Ci/ DNA-bound TraI protein scans for the occurrence of a second mmol) and phage T4 polynucleotide kinase (4). cleavage site at the donor-recipient interface. Cleavage and Site-Specific Recombination of Oligodeoxyri- bonucleotides by TraL Oligodeoxyribonucleotides (5 pmol) were incubated with Tral (4 ,ug, 50 pmol) in 20 ,ul of TNM (20 by bacterial conjugation requires the mM Tris HCl, pH 8.8/50 mM NaCl/5 mM MgCl2) for 3 hr at generation of a defined DNA single strand by a rolling 370C. When appropriate, the reaction was stopped by the circle-type mechanism of replication. Following relaxosome K and formation, the key step in this process is site-specific hydro- addition ofSDS (10 mg/ml) and proteinase (100 ,Ag/ml) lysis of a scissile at oriTby the relaxase, incubation for an additional 30 min at 37°C. Products were which becomes covalently attached to the 5' phosphoryl end analyzed in a 20% (wt/vol) polyacrylamide gel containing 8 ofthe DNA (for review see ref. 1). Covalent adduct formation M urea. Reaction products were quantified by autoradiogra- is thought to conserve the bond energy required for recircu- phy of gels with the storage phosphor technology (5). larization of the transferred strand by a site-specific recom- Sequencing the of TraL Preparation of radio- bination event at the nick site of oriT. Use of the IncPa labeled substrate. Oligodeoxyribonucleotide (17-mer, 5'- plasmid RP4 as a model system for studying the molecular TTCACCTATCCTGCCCG-3', 1 nmol) was labeled at the 3' mechanisms of DNA processing during conjugation showed end with [a-32P]dATP (3.3 pmol; 3000 Ci/mmol). [a-32P]- that all genes required for relaxosome formation are clustered dATP was used instead of [a-32P]ddATP to avoid different adjacent to the intergenic oriT (2). During assembly of RP4 separation properties of labeled and unlabeled oligonucle- relaxosomes, the proteins TraJ, TraI, and TraH interact at otide-peptide adducts during HPLC. Due to the great molar oriT in a cascade-like mechanism preparing the superhelical excess of oligonucleotide over the labeled , the plasmid for initiation of transfer DNA replication (2). The majority of radiolabeled oligonucleotides were 18-mers car- application ofpurified components in vitro revealed that TraJ rying only one 3' [32P]dAMP. The labeled 18-mer was isolated is a specific oriT-binding protein (3) that directs the relaxase by preparative TLC using the United States Biochemical SurePure system. To obtain an amount of oligonucleotide suitable for preparative cleavage by TraI, unlabeled 18-mer The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 2925 Downloaded by guest on October 7, 2021 2926 Biochemistry: Pansegrau et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5'-TTCACCTATCCTGCCCGA-3' was added in 200-fold ex- A cess. Formation of covalent TraI-oligonucleotide adduct. One hundred sixty nanomoles of the mixture of labeled and -18-mer unlabeled 18-mer was incubated with 80 nmol of TraIA3 in 2 = = _-~~~~17-mer by ml TNM for 3 hr at 37°C. The reaction was stopped - d(pCCCG)ddA- addition of SDS (10 mg/ml) and proteinase K (100 ,ug/ml), ...... - 13-mer and incubation was continued for 1 hr. After lyophilization, _PePPOK- the reaction mixture was dissolved in 0.5 ml of water. After centrifugation for 15 min at 20,000 x g and 0WC, the super- natant was applied to a C18 reversed-phase column (4 x 250 a b c d e f (pH mm) equilibrated with 10 mM triethylammonium acetate 5' 5' 5' 3' 3' 3' label 6.5). Peptides were eluted over a period of 3 hr at 0.8 ml/min - + + - + + Tral with a linear gradient of 6-30% acetonitrile in 10 mM trieth- - - + - - + proteinase K ylammonium acetate (pH 6.5). Eluted peptides were detected by A220. Fractions containing pepproK-d(pCCCGA*) were B identified (i) by measuring radioactivity and (ii) by gel n4) electrophoresis. pepPrOK-d(pCCCGA*) was eluted at 18.5% acetonitrile (pepproK means a proteinase K-generated Tral ov peptide with a few amino acid residues; an asterisk indicates n 20 the presence of a [32P]phosphoryl group). One and a half 00z'o 10 vOia nanomoles was obtained. The peak fraction (750 pmol) was 0. analyzed on an Applied Biosystems model 473A sequencer 4) (6). 10 20 RESULTS molar ratio 118-mer]/LTral] of by TraL. Muta- Site-Specific Cleavage Oligonucleotides FIG. 2. Site-specific cleavage of single-stranded oligonucleotides tions in the nick region of plasmid RP4 (Fig. 1) demonstrated by Tral. (A) Tral was incubated with oligonucleotides and reactions that several positions in the 8-bp stretch between the TraJ were stopped and analyzed as described under Materials and Meth- binding region and nic are crucial to Tral-mediated cleavage ods. Presence or absence of Tral in the reactions is shown. Where (7). This indicated that other nucleotides in addition to nic indicated, the cleavage reaction was followed by proteinase K diges- interact with the relaxase. It is expected, therefore, that tion. Oligonucleotides were 32P-labeled at their 3' or 5' end. Positions recognition of oriT sequences by Tral is not exclusively of bands corresponding to the input oligonucleotides [17-mer with mediated by the TraJ-oriT complex. It is also conceivable that 5'-end label, d(p*TTCACCTATCCTGVCCCG), and 18-mer with binding of TraK in this region changes the local DNA 3'-end label, d(TTCACCTATCCTGVCCCG)ddA*] and cleavage that nic is exposed as a single strand for cleavage products [13-mer with 5'-end label, d(p*TTCACCTATCCTG), or topology so pepproK-d(pCCCG)ddA* with 3'-end label] are indicated at right. (B) by Tral (8). Tral was incubated with various amounts of 3'-end-labeled 18-mer as To test the hypothesis of DNA single-strand cleavage, described above. The reactions were stopped by SDS addition and short oligodeoxyribonucleotides spanning the nick region digested with proteinase K. The yield of Tral that could be captured were synthesized and incubated with Tral. The reaction as covalent oligonucleotide adduct is expressed as percentage of the mixture was electrophoresed in a sequencing gel to separate input Tral. the reaction products (Fig. 2A). To visualize the cleavage products derived from both sides of nic, two oligonucleotides 13-mer, which is the expected size for cleavage at nic (Fig. were radioactively labeled at either the 5' or the 3' end. The 2A, lanes b and c). The 3'-labeled counterparts can be oligonucleotides indeed function as substrates for the relax- visualized only after treatment with proteinase K because, in ase because, in addition to the input material, other defined contrast to the covalent TraI-oligonucleotide adduct [Tral- bands were detected. According to our model, cleavage at nic d(pCCCG)ddA*], which does not migrate into the gel (data results in a free 3' hydroxyl end and a covalently attached not shown), pepproK-d(pCCCG)ddA* does (Fig. 2A, lanes e TraI at the 5' nucleotide (9). Reactions containing the 5'- and f). The electrophoretic mobility of the pentanucleotide is labeled substrates yielded an oligonucleotide migrating as a reduced by the remaining amino acid residues. Tral-mediated cleavage of oligonucleotides depends on the presence of nic Mg2+ and has a pH optimum at 8.8 (data not shown). The reaction is essentially indifferent to the concentration of Na+ TCGCGGGTGGGCCTACTTCA XTAT T CCGGCTG or K+. Only at >250 mM NaCl or KCl is cleavage efficiency AGCGCCCACCCGGATGAAGT ATA AC G GGCCGAC reduced significantly. 00 The Covalent Tral-Oligonucleotide Adduct Exists in an j I Equilibrium with Noncleaved Oligonucleotides. To find out TraJ binding region nick region whether or not Tral-mediated oligonucleotide cleavage is a FIG. 1. Organization of the relaxosome assembly region of the reversible process, Tral was incubated with an excess of RP4 oriT. Arrows indicate the positions of inverted sequence re- labeled 18-mer at various molar ratios (Fig. 2B). Plotting the peats. Only the right half of a 19-bp inverted repeat is included. yield ofTral-oligonucleotide adduct against the molar ratio of Deviations from the symmetry are marked by dots within arrows. substrate and reveals that the reaction reaches an Larger dots above and below the sequence designate nucleotides that equilibrium when about 30% of the input TraI exists as are protected against attack by hydroxyl radicals in a TraJ-oriT covalent Tral-oligonucleotide complex. A corresponding re- complex (3). The size of the dots is proportional to the degree of sult is obtained when the 18-mer is incubated with an excess protection. Nucleotides that were identified as important for recog- nition of oriT by Tral (7) are boxed. The extent ofthe nick region and of TraI and the yield of Tral-oligonucleotide adduct is related the TraJ-binding region is indicated by brackets. nic is represented to the input oligonucleotide (data not shown). The latter by a wedge. result indicates that incomplete cleavage is not due to a Downloaded by guest on October 7, 2021 Biochemistry: Pansegrau et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2927

partially inactive Tral preparation but is a property of the reaction itself. A 6-nt Sequence Is the Core Region Required for the -30-mer TraI-Mediated Cleaving-Joining Reaction. To define the se- - - 26-mer quence requirements for the cleaving-joining reaction, we employed a set of synthetic 22-mer oligonucleotides contain- ing single base exchanges in the nick region. The functional - 22-mer boundaries of the nick region were previously identified by testing hydroxylamine mutants of oriT in vivo and in vitro (7). ~4UI __ .- 18-mer The mutant oligonucleotides contained preferentially trans- versions because these cannot be obtained by hydroxylamine mutagenesis (Fig. 3). An oligonucleotide in which the G at nic (GVC) is replaced by A is not cleaved. This result demon- a b c d e strates the high specificity of the cleavage reaction and - + + + + Tral confirms the data obtained with oriT mutant DNA described + + + + - Mg2+ 6 nt located upstream nic + + + - + 30-mer* previously (7). Essentially, of - - - + - 29-mer appear to be critical for recognition by Tral, whereas down- + + - + + 18-mer* stream of nic no base specificity could be found within the - - + -- 17-mer first 2 nt (Fig. 3). B Tral Catalyzes Site-Specific Recombination/Joining of Sin- nic gle-Stranded Oligodeoxyribonucleotides. oriT sites are subject 30-m er to site-specific recombination dependent on the presence of 18-mer transfer gene products (for review see ref. 1). To see whether Tral can join fragments cleaved from two distinguishable 1[Tral, Mg2+ partners, we modified our standard reaction by offering two 26-mer oligonucleotides of different lengths (30- and 18-mer, Fig. 4). W ~~~~22-mer Either both or only one of the oligonucleotides was labeled at FIG. 4. Site-specific recombination ofoligonucleotides catalyzed the 3' end in order to define the origins ofthe hybrid products. by Tral. (A) Tral was incubated with the indicated oligonucleotides. Both ofthe expected hybrid oligonucleotides (26- and 22-mer) After 2 hr, reactions were stopped by the addition of SDS and were generated, indicating that the cleaving-joining reaction analyzed electrophoretically. Presence or absence of Tral, Mg2+, catalyzes site-specific recombination in vitro. and oligonucleotides in the reaction mixtures is indicated. Oligonu- TraI-d(pCCCG)ddA* Transfers Its DNA Moiety to a Pre- cleotides that were 32P-labeled at their 3' ends are marked with an formed nic 3' Terminus. To find out whether a preformed nic asterisk. The terms 17-mer and 23-mer refer to the corresponding 3' terminus can act as an acceptor of oligonucleotide frag- unlabeled oligonucleotides. Positions of bands corresponding to the ments, 3'-end-labeled 30-mer was incubated with Tral in a input oligonucleotides [30-mer, d(GGGCCTACTTCACCTATCCT modified recombination reaction. Following a preincubation GVCCCGGCTG)ddA*, and 18-mer; see Fig. 2] and recombination products (22-mer and 26-mer) are indicated at right. (B) Schematic step, a 13-mer oligonucleotide covering the Tral recognition representation of the Tral-mediated recombination of oligonucleo- region but ending at the nic 3' terminus was added at various tides at nic. molar ratios (Fig. SA, lanes a-f). A 22-mer product demon- strates that the 13-mer participates in the recombination Peptide Mapping of the Covalent Attachment Site of TraI to reaction either by displacing the 5' end of the 30-mer com- the 5'-Terminal Nucleotide at the Cleavage Site. To locate the plexed with Tral or by reaction with free TraI-d(pCCCGGC- center of the catalytic site of Tral we analyzed oligonucleo- TG)ddA*, which might exist under the conditions employed. tide cleavage reaction products that were digested with Quantitative evaluation of the reaction partners (Fig. 5B) trypsin (Fig. 6A, lanes b-d), Glu-C (lanes e-g), Lys-C (lanes revealed that (i) even at moderate molar ratios of 13-mer to h-j), or proteinase K (lanes k-m) to generate small peptides 30-mer an efficient transfer of the 30-mer 3'-terminal part to susceptible to separation in sequencing gels. Three of the the 13-mer takes place and (ii) addition of the 13-mer de- proteases used (trypsin, Glu-C, and Lys-C) yielded peptide- creases the yield of Tral-oligonucleotide adduct by shifting oligonucleotide adducts with identical electrophoretic mobil- the cleavage equilibrium to the joined-oligonucleotide form. ity (Fig. 6A). Inspection of the amino acid sequence of Tral Tral recognition revealed that Tyr22 would be located on peptides generated by the three proteases, which have not only very similar C T A T C C T GvC C molecular weights but also identical charges (Fig. 6B). Since A 1.2 0.9 N.D. X 0.8 N.D. X 1.3 1.0 this situation is unique within the Tral sequence, we tenta-

C - 1.3 0.5 N.D. * * N.D. X * tively assigned the Tral active site to Tyr22. This assumption G N.D. 1.2 0.1 X N.D. X X - 1.0 N.D. was supported by the finding that the Tyr22 -* Phe mutant Tral is in terms T N.D. _ 0.3 - N.D. N.D. 0 1.1 1.0 N.D. protein inactive ofDNA cleavage and that the proteinase K-generated peptide-oligonucleotide adduct of FIG. 3. Cleavage efficiencies of altered oligodeoxyribonucle- another Tral mutant (Thr24 -> Ala) has an altered electro- otides encompassing the nick region. Oligonucleotides labeled at phoretic mobility as compared to the wild-type peptide their 3' ends were incubated under standard conditions with Tral. (unpublished work). Reactions were stopped by the addition of SDS and proteinase K. Peptide Sequencing of Oligonucleotide-Bound Tral Reveals Products were quantified as described under Materials and Methods Tyr22 as the Site of Covalent Attachment. Preliminary sequenc- and cleavage efficiencies were normalized to the unaltered oligonu- ing experiments using Tral peptides covalently attached to cleotide [22-mer, d(ACTTCACCTATCCTGVCCCGGC)ddA*]. Nu- nicked oriT plasmid DNA indicated that the very N-terminal cleotide exchanges in certain positions of the 22-mer are related to part of the forms the This the bases listed at left. X, no cleavage product was detectable (<0.02 protein phosphodiester linkage. of the cleavage efficiency with the unaltered 22-mer); N.D., not result is in agreement with the finding that the smallest soluble determined. A bracket marks nucleotide positions that are important deletion derivative of Tral truncated at the C terminus for recognition of the oligonucleotide by Tral. The cleavage site is (TraIA3, 417 amino acids; ref. 10) is fully active in the indicated (V). cleaving-joining reaction with plasmid DNA as well as with Downloaded by guest on October 7, 2021 2928 Biochemistry: Pansegrau et al. Proc. Natl. Acad. Sci. USA 90 (1993) A

- -~~~~~~~~18-mer_s_s__ _ 1 ~~~pep- 30-mer IMIW .W-11, _ ~~__ = g J ~~~d(PCCCG)ddA' INW '-W-,,. -,.. "F.".

~--- -22-mer a b c d e f g h i j k I m pepproK --' - - - - -d(pCCCG GCTG)ddA* - Tryp Glu-C Lys-C ProK a b c d e f g B 5 10 15 20 25 30 ~~~~~_ 1 3 m e r T T TT MAIAVPMRSIKKSOFA,LVY TDEPGYTYER.... B 10-5 Tryp v Glu-C U i Ef3 @ l Lys-C 0 E FIG. 6. Mapping ofoligonucleotide-associated peptides by diges- 0 tion of TraI-d(pCCCG)ddA* with specific proteases. (A) The 3'-end- labeled 18-mer oligonucleotide was incubated with Tral. Reactions were terminated by addition ofproteases together with the respective compounds: trypsin, 10 mM CaCl2, 0.1% SDS; endoproteinase Glu-C, 10 mM CaCl2, 0.1% SDS; endoproteinase Lys-C, 0.1% SDS; 0 5 10 proteinase K, 1% SDS. Lanes: a, protease omitted; b-d, trypsin (50, molar ratio [1 3-mer]/[30-mer] 100, and 200 ng/,l, respectively); e-g, endoproteinase Glu-C (20, 200, and 2000 ng/,u); h-j, endoproteinase Lys-C (5, 10, and 20 FIG. 5. Reaction of covalent Tral-oligonucleotide adduct with a ng/,ul); k-m, proteinase K (10, 100, and 1000 ng/ml). Positions of preformed 3' terminus. (A) Tral (80 pmol) was incubated for 1 hr bands corresponding to the input 18-mer and to Tral peptides under standard conditions with 3'-end-labeled 30-mer (16 pmol, see covalently linked to d(pCCCG)ddA* are marked at right. (B) Cleav- Fig. 4), followed by addition ofvarious amounts ofunlabeled 13-mer, age sites of various proteases are symbolized above and below the d(TTCACCTATCCTG). Incubation was continued for 2 hr before N-terminal amino acid sequence of Tral. Peptides containing the the reactions were stopped. Lanes: a, 4 pmol of 13-mer; b, 8 pmol; active-site tyrosine (shaded box) are indicated by bars. Positions of c, 16 pmol; d, 32 pmol; e, 80 pmol; f, 160 pmol; g, 13-mer omitted. charged amino acid residues are marked by 3 or E. Positions of the bands corresponding to the input oligonucleotide (30-mer) and the products ofthe transfer reaction (22-mer) and ofthe cleavage reaction are indicated at right. (B) Reaction products shown second-cleavage model of DNA transfer termination (1). in A were quantified and the radioactivity found in each band was However, another possible hypothesis, supported by the plotted against the molar ratio of the input oligonucleotides. *, finding that a preformed nic 3' terminus can function as pepproK-d(pCCCGGCTG)ddA*; e, 30-mer; o, 22-mer. acceptor for the TraI-catalyzed joining reaction (Fig. 5), is oligonucleotides (data not shown). To demonstrate the site of D E covalent attachment directly, we isolated the proteinase 0.06 K-generated peptide-oligonucleotide adduct by preparative 0.3 0.06 o l E HPLC. Since after reaction of Tral with oligonucleotide only 0.2 a minor fraction of the input protein can be found covalently A attached to DNA (see above), we used TraIA3 to improve the 0.04 tI iJ 0.1 relative abundance of the desired peptide-oligonucleotide complex. N-terminal sequencing of pepPrOK-d(pCCCGA*) re- 0 . JI.L 4 8 12 16 20 2428 4 8 12 16 20 24 28 vealed that following proteinase K treatment a tetrapeptide < e.-AI having the sequence Lys-Xaa-Ile-Thr, corresponding to amino acids 21-24 of Tral, remained covalently attached to the 0.3 0.3- pentanucleotide (Fig. 7). In the second degradation cycle (Fig. 7III) no significant signal was found. This position corre- 0.2 0.2- A sponds to Tyr22, which apparently constitutes the linkage 0.1 0.1 between Tral and the DNA and therefore escapes detection. T

0 1 a,. 4 8 12 16 20 24 28 4 8 12 16 20 24 28 DISCUSSION retention time [min] Models for the mechanism of DNA transfer are conjugative FIG. 7. HPLC traces from N-terminal sequence analysis of based on the rolling-circle mode of DNA replication mainly proteinase K-generated Tral peptide covalently attached to oligo- because a DNA single strand is transferred unidirectionally nucleotide. pepproK-d(pCCCGA*) (750 pmol) was isolated by HPLC from donor to recipient cells (11). We present evidence that and sequenced. Phenylthiohydantoin amino acid derivatives were the RP4 relaxase catalyzes functions required for initiation detected by measuring the absorbance at 220 nm. (I) Standard and termination of rolling-circle replication. The 5' phospho- mixture of phenylthiohydantoin amino acid derivatives (60 pmol ryl terminus of the strand destined to be transferred is each); the peak labeled with A was assigned to diphenylthiourea, a covalently attached to Tyr22 ofTral. During the side product that usually forms during Edman degradation. (II-VI) transmission, Cycles 1-5 of Edman degradation of peppr0K-d(pCCCGA*). III-VI strand being transferred is constantly scanned for the recon- are difference plots that were obtained by subtracting the trace ofthe stituted nick-region sequence. A transesterification occurs preceding cycle from the actual trace. Assignments of the phenyl- when the sequence is recognized by TraI to yield a closed thiohydantoin derivatives yielding the sequence Lys-Xaa-Ile-Thr circular DNA molecule (Fig. 8A). Our results agree with the (KXIT) are indicated. Downloaded by guest on October 7, 2021 Biochemistry: Pansegrau et al. Proc. Natl. Acad. Sci. USA 90 (1993) 2929

Tral cleaving-joining reactions can be performed on synthetic A oligonucleotides containing the lower strand of Ti border sequences, as well as on RP4 nick-region oligonucleotides (W.P., F. Schoumacher, B. Hohn, and E.L., unpublished T_recognition/ work). These results are paralleled by the finding that cognate relaxases share motifs possibly involved in specific recognition of single-stranded DNA or in the -_L/f \~~_1 9-bp inverted catalytic activity of the (ref. 12; W.P. and E.L., unpublished work). Sequence similarities found in various 5' a srong interactio relaxases as well as in their target sequences strongly suggest that many different DNA transfer systems have evolved from a common ancestor and that therefore the mechanistic details discussed here are generally applicable. The finding that the active-site Tyr22 of RP4 Tral corresponds to Tyr29 of pTiA6 VirD2, the presence of which was shown to be essential for in vivo nicking of border sequences (13), confirms the hy- pothesis that bacterial conjugation and T-DNA transfer to plants are analogous processes. FIG. 8. Alternative models for termination of transfer DNA replication by transesterification reactions. We are grateful to Heinz Schuster for generous support and stimulating discussions. We appreciate the constructive comments that a unit-length transfer strand is transmitted without and critical reading ofthe manuscript by Ellen L. Zechner. This work extension at the 3' hydroxyl end (Fig. 8B). This mechanism was financially supported by Sonderforschungsbereich Grant would require a priming reaction on the retained strand to 344/B2 of the Deutsche Forschungsgemeinschaft to E.L. restore the double-stranded plasmid in the donor . Apparently, cleavage and rejoining of oligonucleotides in 1. Wilkins, B. & Lanka, E. (1993) in Bacterial Conjugation, ed. Clewell, D. B. (Plenum, New York), pp. 105-136. vitro are dependent on the presence of a 6-nt sequence. Yet, 2. Pansegrau, W., Balzer, D., Kruft, V., Lurz, R. & Lanka, E. even within this short sequence, some base exchanges are (1990) Proc. Natl. Acad. Sci. USA 87, 6555-6559. allowed without loss of activity. Since nucleotide sequences 3. Ziegelin, G., Furste, J. P. & Lanka, E. (1989) J. Biol. Chem. which would fulfill the requirements for in vitro cleavage and 264, 11989-11994. rejoining are present several times on RP4, further signals 4. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular that increase the specificity of the termination reaction must Cloning: A Laboratory Manual (Cold Spring Harbor Lab., exist. A possible candidate for this function is the 19-bp Plainview, NY). 5. Johnston, R. F., Pickett, S. C. & Barker, D. L. (1990) Elec- inverted repeat sequence adjacent to the nick region, only the trophoresis 11, 355-360. right half of which is needed for relaxosome formation. The 6. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, hairpin structure that might form on single-stranded DNA W. J. (1981) J. Biol. Chem. 256, 7990-7997. could function as a pausing signal for DNA transfer, enabling 7. Waters, V. L., Hirata, K. H., Pansegrau, W., Lanka, E. & Tral to execute the recircularization reaction. Guiney, D. G. (1991) Proc. Natl. Acad. Sci. USA 88, 1456- Recently, sequence comparison studies with the IncP nick 1460. region revealed a striking nucleotide sequence similarity to 8. Ziegelin, G., Pansegrau, W., Lurz, R. & Lanka, E. (1992) J. the nick regions of several other transmissible plasmids, Biol. Chem. 267, 17279-17286. 9. Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. including the border sequences of agrobacterial Ti and Ri 265, 10635-10644. plasmids (7, 12). Our experiments testing oligonucleotides 10. Furste, J. P., Pansegrau, W., Ziegelin, G., Kroger, M. & containing single base exchanges (Fig. 3) suggest that all the Lanka, E. (1989) Proc. Natl. Acad. Sci. USA 86, 1771-1775. nick and border regions listed in ref. 12 might function as 11. Ohki, M. & Tomizawa, J.-I. (1968) Cold Spring Harbor Symp. substrates in the single-stranded-DNA cleaving-joining re- Quant. Biol. 33, 651-658. action catalyzed by RP4 TraI. Preliminary experiments using 12. Pansegrau, W. & Lanka, E. (1991) Nucleic Acids Res. 19, 3455. purified VirD2 protein have indeed demonstrated that similar 13. Vogel, A. M. & Das, A. (1992) J. Bacteriol. 174, 303-308. Downloaded by guest on October 7, 2021