Advertisement

Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site*

  • María Lucas
    Footnotes
    Affiliations
    Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-UC-IDICAN, C. Herrera Oria s/n, 39011 Santander
    Search for articles by this author
  • Blanca González-Pérez
    Footnotes
    Affiliations
    Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-UC-IDICAN, C. Herrera Oria s/n, 39011 Santander
    Search for articles by this author
  • Matilde Cabezas
    Affiliations
    Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-UC-IDICAN, C. Herrera Oria s/n, 39011 Santander
    Search for articles by this author
  • Gabriel Moncalian
    Affiliations
    Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-UC-IDICAN, C. Herrera Oria s/n, 39011 Santander
    Search for articles by this author
  • Germán Rivas
    Affiliations
    Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain
    Search for articles by this author
  • Fernando de la Cruz
    Correspondence
    To whom correspondence should be addressed. Tel.:34-942-201942; Fax:34-942-201945;
    Affiliations
    Departamento de Biología Molecular, Universidad de Cantabria, Instituto de Biomedicina y Biotecnología de Cantabria, Consejo Superior de Investigaciones Científicas-UC-IDICAN, C. Herrera Oria s/n, 39011 Santander
    Search for articles by this author
  • Author Footnotes
    * This work was supported by Spanish Ministry of Education Grants BFU2008-00995/BMC and CIT-010000-2008-4.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
    1 Recipient of a predoctoral fellowship from the Public Foundation “Marqués de Valdecilla.” Present address: Dept. of Chemistry and Biochemistry, Gene Center, University of Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany.
    2 Present address: Dept. of Biochemistry, University of Cambridge, 80 Tennis Court Rd., Cambridge CB2 1GA, United Kingdom.
Open AccessPublished:January 08, 2010DOI:https://doi.org/10.1074/jbc.M109.057539
      TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

      Introduction

      Bacterial conjugation is an efficient and sophisticated DNA transport mechanism, genetically encoded by self-transmissible plasmids. The transfer of DNA by bacterial conjugation plays an important role in the genetic variability of bacteria as well as in the propagation of antibiotic resistance and virulence factors (
      • de la Cruz F.
      • Davies J.
      ). In order to avoid the spread of antibiotic resistance genes via bacterial conjugation, one promising strategy is the use of anti-conjugation-based antimicrobial agents (
      • Filutowicz M.
      • Burgess R.
      • Gamelli R.L.
      • Heinemann J.A.
      • Kurenbach B.
      • Rakowski S.A.
      • Shankar R.
      ,
      • Potts R.G.
      • Lujan S.A.
      • Redinbo M.R.
      ). Our group identified unsaturated fatty acids as conjugation inhibitors (
      • Fernandez-Lopez R.
      • Machón C.
      • Longshaw C.M.
      • Martin S.
      • Molin S.
      • Zechner E.L.
      • Espinosa M.
      • Lanka E.
      • de la Cruz F.
      ). Their target is unknown, although membrane-associated ATPases could be good candidates. Because the relaxase is the key catalytic enzyme in the conjugative process, it is, a priori, a better target for a specific inhibitor. Potts et al. (
      • Lujan S.A.
      • Guogas L.M.
      • Ragonese H.
      • Matson S.W.
      • Redinbo M.R.
      ) found that bisphosphonates inhibited the activity of plasmid F relaxase TraI. Their effect on conjugation inhibition was small, although, surprisingly, they could specifically kill relaxase-containing cells. Moreover, bacterial relaxases might find a use as tools for site-specific DNA delivery to target eukaryotic cells for gene therapy (
      • Llosa M.
      • de la Cruz F.
      ). Thus, a detailed study of the specificity determinants of the reaction performed by relaxases could lead to the a la carte design of relaxases able to act on any potentially interesting sequence (
      • González-Pérez B.
      • Carballeira J.D.
      • Moncalián G.
      • de la Cruz F.
      ).
      Conjugative DNA processing is carried out by the relaxosome, composed by the enzyme relaxase and auxiliary proteins that act on the oriT region (see Ref.
      • Zechner E.L.
      • de la Cruz F.
      • Eisenbrandt R.
      • Grahn A.M.
      • Koraimann G.
      • Lanka E.
      • Muth G.
      • Pansegrau W.
      • Thomas C.M.
      • Wilkins B.M.
      • Zatyka M.
      for a review). It starts by a site- and strand-specific DNA cleavage reaction that occurs at a specific oriT site called nic. The nic cleavage reaction is mediated by a tyrosine residue that catalyzes a transesterification reaction. After cleavage, the relaxase remains covalently bound to the 5′-end of the cleaved strand via a phosphotyrosyl linkage, whereas the 3′-hydroxyl is sequestered by tight non-covalent interaction with the relaxase. The cleavage reaction is reversible because the free DNA 3′-hydroxyl group can attack the 5′-phosphotyrosyl bond. However, when the relaxase-DNA complex releases the 3′-OH portion of the DNA (as when it is transported to the recipient cell), a second tyrosine can attack a second nic site positioned at the protein active site. This type of reaction takes place at the end of conjugation for regenerating the oriT in the recipient cell, and it is known as strand transfer reaction (
      • Gonzalez-Perez B.
      • Lucas M.
      • Cooke L.A.
      • Vyle J.S.
      • de la Cruz F.
      • Moncalián G.
      ,
      • Garcillán-Barcia M.P.
      • Jurado P.
      • González-Pérez B.
      • Moncalián G.
      • Fernández L.A.
      • de la Cruz F.
      ).
      TrwC is a multidomain protein of 966 amino acids that forms dimers in solution (
      • Grandoso G.
      • Llosa M.
      • Zabala J.C.
      • de la Cruz F.
      ). The N-terminal part of the protein contains the relaxase domain (amino acids 1–300) (
      • Grandoso G.
      • Avila P.
      • Cayón A.
      • Hernando M.A.
      • Llosa M.
      • de la Cruz F.
      ), whereas the C-terminal region (amino acids 192–966) is responsible for dimerization and DNA-helicase activity, required for unwinding the transferring DNA (
      • Llosa M.
      • Bolland S.
      • Grandoso G.
      • de la Cruz F.
      ,
      • Llosa M.
      • Grandoso G.
      • Hernando M.A.
      • de la Cruz F.
      ). TrwC specifically nicks oriT-containing supercoiled plasmids in vitro in the absence of accessory proteins and remains covalently bound to the 5′-end of the cleaved DNA strand (
      • Llosa M.
      • Grandoso G.
      • de la Cruz F.
      ). The nicking activity of TrwC allows intermolecular site-specific recombination between two plasmids containing oriT in the absence of conjugation (
      • Llosa M.
      • Bolland S.
      • Grandoso G.
      • de la Cruz F.
      ). Two specific tyrosyl residues in TrwC, Tyr18 and Tyr26, are involved in the DNA strand transfer reactions (
      • Gonzalez-Perez B.
      • Lucas M.
      • Cooke L.A.
      • Vyle J.S.
      • de la Cruz F.
      • Moncalián G.
      ,
      • Garcillán-Barcia M.P.
      • Jurado P.
      • González-Pérez B.
      • Moncalián G.
      • Fernández L.A.
      • de la Cruz F.
      ,
      • Grandoso G.
      • Avila P.
      • Cayón A.
      • Hernando M.A.
      • Llosa M.
      • de la Cruz F.
      ). Tyr18 catalyzes the first strand cleavage, whereas Tyr26 is involved in the strand transfer reaction that terminates the DNA processing. Between these two steps in conjugation, the DNA strand that was first cleaved is displaced by the helicase activity of TrwC. Similar reactions occur during processing of F plasmid oriT by the related relaxase TraI_F. The relaxases of F and R100 plasmids also act as bifunctional relaxases, with relaxase and helicase domains in the same protein (
      • Reygers U.
      • Wessel R.
      • Müller H.
      • Hoffmann-Berling H.
      ,
      • Matson S.W.
      ,
      • Fukuda H.
      • Ohtsubo E.
      ).
      Conjugative and mobilizable plasmids of the same MOB family show conservation of the DNA sequence of oriT (
      • Garcillán-Barcia M.P.
      • Francia M.V.
      • de la Cruz F.
      ,
      • Francia M.V.
      • Varsaki A.
      • Garcillán-Barcia M.P.
      • Latorre A.
      • Drainas C.
      • de la Cruz F.
      ). Nevertheless, the oriT sequences specifically involved in the so-called initiation and/or termination reactions are unknown for the vast majority of plasmids. The initiation reaction is the first cleavage reaction performed by Tyr18 in TrwC. The termination reaction is the second cleavage and strand transfer reaction performed by Tyr26 in TrwC. In most analyzed oriT regions, an inverted repeat (IR, named IR2 in R388) is located upstream the nic site (
      • Francia M.V.
      • Varsaki A.
      • Garcillán-Barcia M.P.
      • Latorre A.
      • Drainas C.
      • de la Cruz F.
      ,
      • Parker C.
      • Becker E.
      • Zhang X.
      • Jandle S.
      • Meyer R.
      ), which is recognized either by the relaxase or by some auxiliary relaxosomal protein (
      • Zechner E.L.
      • de la Cruz F.
      • Eisenbrandt R.
      • Grahn A.M.
      • Koraimann G.
      • Lanka E.
      • Muth G.
      • Pansegrau W.
      • Thomas C.M.
      • Wilkins B.M.
      • Zatyka M.
      ). The proximal arm of the IR and the region surrounding the nic site are sufficient for the initiation reaction in plasmids R64 and R1162, whereas a larger DNA substrate that includes the complete IR is required in the termination reaction. Conversely, in F plasmid, initiation demands a larger DNA substrate than the termination reaction (
      • Gao Q.
      • Luo Y.
      • Deonier R.C.
      ).
      The three-dimensional crystal structure of the relaxase domain of TrwC (TrwCR) has been solved in complex with its cognate 25-base oligonucleotide substrate, folded in a DNA hairpin (
      • Guasch A.
      • Lucas M.
      • Moncalián G.
      • Cabezas M.
      • Pérez-Luque R.
      • Gomis-Rüth F.X.
      • de la Cruz F.
      • Coll M.
      ). The DNA is firmly held by the relaxase by two identifiable binding sites. The hairpin forms an almost perfect B-DNA that is bound by two different motifs through its major and minor grooves. The nic-proximal ssDNA
      The abbreviations used are: scDNA
      supercoiled DNA
      ssDNA
      single-stranded DNA
      dsDNA
      double-stranded DNA.
      is housed in a deep narrow cleft that contains the relaxase catalytic site. Nucleotides involved in that “frozen” interaction with the relaxase were established, but the three-dimensional structure could not reveal which nucleotides participate in the enzymatic reactions of cleavage and strand transfer. In this work, we characterize the biochemical and biophysical properties of the TrwC-DNA complex. In addition, we study the elements involved in DNA sequence recognition in the independent reactions catalyzed by TrwC during conjugative DNA processing. We present evidence that TrwC recognizes its target nic region in two steps: an initial scDNA binding involving the proximal arm of IR2, followed by recognition of the adjacent ssDNA binding site that situates the cleavage site in the right position to be cleaved.

      DISCUSSION

      The interaction between a conjugative relaxase and its target site is the initial step for conjugative DNA processing. Recognition of the nic site has to be specific enough so that a single sequence can be selected out of a complete bacterial genome (in fact out of a number of genomes of potential bacterial hosts). As we show in this paper, this exquisite recognition is brought about by separating it into two different steps. TrwC binds to a palindromic DNA sequence formed in a double-stranded region of the DNA (binding sequence) and then cleaves in an adjacent sequence if a second specific sequence is found (cleavage sequence). TrwC binding to the palindromic sequence IR2 was previously defined by protein crystallography. The present results indicate that TrwC binds IR2 with high affinity. Moreover, the stoichiometry of the complex was found to be a 1:1 molar ratio. This oligomerization state is consistent with the data presented in Ref.
      • Gonzalez-Perez B.
      • Lucas M.
      • Cooke L.A.
      • Vyle J.S.
      • de la Cruz F.
      • Moncalián G.
      . Although this perfect palindromic IR was recognized and bound by TrwC with high affinity, shorter oligonucleotides not containing the entire IR were effectively cleaved by TrwC.
      When binding and cleavage of oligonucleotides R(25 + 4), R(14 + 4), R(12 + 4), and R(6 + 4) were compared, we observed that the absence of the distal repeat of the IR2 deteriorated TrwC binding ability (Fig. 1). However, nic cleavage activity remained intact in the oligonucleotides without the IR2 distal arm, indicating that IR2 is dispensable for cleavage but essential for high affinity binding to the relaxase. The relaxase binds these oligonucleotides poorly but sufficiently well to recognize the sequence required for nic cleavage. These results suggest that TrwCR has to recognize one sequence for binding and another for nic cleavage, although both are required for proper binding, and both are required for a proper nic cleavage.
      nic cleavage efficiency was increased by reduction of the length of the sequence located 5′ of the cleavage site (from 25 to 12 nucleotides). In the same way, we observed an inverse relationship between binding and nic cleavage efficiency. This apparent contradiction was explained by experiments using suicide nucleotides (
      • Gonzalez-Perez B.
      • Lucas M.
      • Cooke L.A.
      • Vyle J.S.
      • de la Cruz F.
      • Moncalián G.
      ). These nucleotides displaced the reaction equilibrium to the formation of products, therefore reducing the reverse joining reaction. In this way, R(25s + 4) did not show reduced nic cleavage activity but rather increased rejoining efficiency, due to better TrwC binding that positions the 3′-OH in a better place to attack the phospho-tyrosyl bond and religate the oligonucleotide. In the same line of thought, we observed that increasing the incubation time produced higher nic cleavage yields in all cases. In fact, after 48 h of incubation, all oligonucleotides were cleaved to a similar amount. Therefore, different cleavage yields are due to the different dissociation rates of the cleaved product and not to different recognition or cleavage efficiency. Unstable binding could provoke dissociation of the 5′ product that normally remains captured by the relaxase. Consequently, the equilibrium of the cleavage-joining reaction would be displaced toward the nic cleavage products.
      To further analyze the role of the different DNA residues in TrwC binding and cleavage, we performed mutagenesis analysis, the results of which are summarized on Fig. 3. According to these results, we can dissect the TrwC binding site in two regions: the IR2 binding site (comprising the distal and proximal arms) and the single-stranded binding site.

      IR2 Distal Arm

      As mentioned above, IR2 is essential for oligonucleotide binding but not for scDNA cleavage. Thus, mutations in the distal arm, which affect ssDNA but not scDNA binding, only slightly affect mobilization. As expected, binding of the oligonucleotide containing this mutation is impaired but not its cleavage. Strikingly, the mobilizable scDNA was cleaved by TrwC with the same efficiency as wild type oriT. These results are surprising, considering that the DNA sequence bound by TrwC starts at −25 according to the three-dimensional structure of the TrwC-nic complex. Thus, it seems that the role of the IR2 distal arm is to allow cruciform formation (that probably only occurs during the termination reaction on the transported T-strand), because specific interactions with TrwC do not play a crucial role.

      IR2 Loop

      Mutations in the IR2 loop did not affect substantially any of the properties analyzed (see Rm8–11 results in Fig. 3). This is consistent with TrwC-nic crystal structure, where no direct interaction between TrwC and any of the four nucleotides of the loop was observed.

      IR2 Proximal Arm

      This segment is essential for mobilization, binding, and cleavage of scDNA (but not ssDNA cleavage). The specific interactions of TrwC with these residues are abundant in the crystal structure. Thus, modification of these residues abrogates TrwCR binding to this site. TrwCR recognizes not only the B-DNA form of IR2 (i.e. its proximal arm on dsDNA) but also the nitrogenated bases of the nucleotides forming the IR, as observed in the mutant that changes the nucleotides but maintains an IR at the same position as IR2. In this case, mobilization and binding activity are both lost. Because the specific sequence of the distal arm or the loop is not essential, but the specific sequence of the hairpin is essential, we can conclude that the interactions of this DNA region with the protein are crucial in the recognition.
      These data allow us to present a model for the role of IR2 in R388 conjugation (Fig. 5). According to this model, TrwC recognizes the dsDNA containing the proximal arm of IR2 in the donor cell (Fig. 5A). This is consistent with the fact that TrwC recognizes and cleaves scDNA containing mutations in the IR2 distal arm. It is also consistent with the crystal structure of the TrwC-nic complex if we understand that the hairpin bound in the structure is a representation of the proximal arm dsDNA bound by the relaxase in vivo. In fact, the absence of involvement of the loop in recognition makes a single-stranded cruciform containing the distal and proximal arms of IR2 indistinguishable from a scDNA containing both strands of the proximal arm. High affinity binding to the proximal arm allows local melting of the DNA around the cleavage site and the generation of a U-shaped turn in the transferred ssDNA strand that positions the nic site in the TrwC active site (Fig. 5B). The specific requirements of the nucleotides that form the U-shaped turn will be discussed below. After cleavage, the displaced ssDNA in the donor DNA molecule is transported to the recipient cell being piloted by the relaxase, where the ssDNA is recircularized. In this step, the reaction requires TrwC to recognize the nic site after one round of replication. However, because the DNA is transported in a single-stranded form, the new binding site will not be dsDNA this time but ssDNA. It is in this second recognition step that both arms of the IR2 are needed (Fig. 5C).
      Figure thumbnail gr5
      FIGURE 5Model of TrwC oriT recognition in the conjugation process. A, TrwC (light shaded element) recognizes the dsDNA containing the proximal arm of IR2 (DNA in sticks representation) in the donor cell. B, high affinity binding to the proximal arm allows local melting of the DNA around the cleavage site and the generation of a U-shaped turn in the transferred ssDNA strand that positions the nic site in the TrwC active site. C, TrwC recognizes the ssDNA containing both arms of IR2 in the recipient cell. Red, bases T20–T25, which are recognized in the ssDNA processing; cyan, additional bases (G12–A19) relevant for scDNA relaxation; dark green, complementary sequence of the proximal arm of IR2; yellow, IR2 loop; light green, distal arm of IR2; orange, DNA generated by rolling circle replication. The position of TrwC Tyr18 is indicated in magenta on the protein surface. The sequence of the dsDNA or ssDNA recognized by TrwC is shown below with the same color code as in the model.
      Analogous results were observed for plasmid R1162 (
      • Becker E.C.
      • Meyer R.J.
      ), where it was found that mutations in the outer arm of the IR adjacent to nic did not affect mobilization. These authors reported that this part of nic was involved in the termination reaction.
      An interesting result was obtained with the mutants in G17. This nucleotide should interact with its counterpart C2. Instead, according to the available crystal structures, G17 interacts with TrwC residues Arg81 and Asp183. Due to this interaction, G17 is the first nucleotide of the ssDNA region, and it seems that the interaction of G17 with Arg81 and Asp183 is essential for the extension of the ssDNA segment up to the nic site. This structural observation could explain why the mutant oligonucleotide is bound and cleaved by the protein, but nevertheless the corresponding plasmid cannot be mobilized.

      Single-stranded Binding Site

      Using oligonucleotides lacking IR2 (R(14 + 4), R(12 + 4), and R(6 + 4)), we observed that IR2 is dispensable for cleavage but essential for high affinity binding to the relaxase (Fig. 1). The relaxase binds the above oligonucleotides poorly but sufficiently to recognize and cleave the nic site. Even oligonucleotide R(6 + 4) seemed to contain enough sequence information to position the scissible phosphate in the catalytic center so that the oligonucleotide could be cleaved.
      As observed when binding to oligonucleotides R(25-6), R(25-3), and R(25-0) was compared, the ssDNA binding site also contributes to TrwC stable binding (
      • Guasch A.
      • Lucas M.
      • Moncalián G.
      • Cabezas M.
      • Pérez-Luque R.
      • Gomis-Rüth F.X.
      • de la Cruz F.
      • Coll M.
      ). These results suggest that TrwCR is recognizing two different sequences, one for high affinity binding and a second one for nic cleavage.
      The effect of the mutations between IR2 and the nic cleavage site corresponded to what could have been predicted from the crystal structure. Inside this core region (nucleotide positions 13–27), two phenotypes could be distinguished. Mutations in the segment from position 20 to 27 resulted in oligonucleotides inactive for cleavage. Nucleotides 20–27 form the U-shaped turn necessary to localize the nic site at the catalytic center. Mutations in any of these nucleotides affect the interaction with several residues within the TrwCR cleft, where the U turn is bound. Moreover, the base interaction between T25 and G22 stabilizes the U-turn that drives the nic site to the close proximity of the catalytic tyrosine. This three-base intrastrand interaction to form the U-turn was also observed in the crystal structure of the TraI relaxase (
      • Datta S.
      • Larkin C.
      • Schildbach J.F.
      ,
      • Hekman K.
      • Guja K.
      • Larkin C.
      • Schildbach J.F.
      ).
      On the other hand, mutations in the region from 19 to 13 resulted in oligonucleotides that were cleaved with enhanced efficiency. A similar result occurred when oligonucleotide R(12 + 18) was used, suggesting that the lack of appropriate interactions in this region could be affecting (i) the stability of the bound oligonucleotide and thus its off-rate (unlikely because Kd is not grossly affected, and complex half-life is 11 h) or (ii) the positioning of the oligonucleotide with respect to the cleavage site. Perhaps binding to this region is modulating the cleavage efficiency of the protein. In fact, Williams and Schildbach (
      • Williams S.L.
      • Schildbach J.F.
      ) also found that similar mutations in the nic site of plasmid F resulted in enhanced cleavage at high relaxase concentration.
      In summary, TrwC recognizes dsDNA and specifically binds the proximal arm of IR2. Upon binding, the bound DNA is distorted so that local DNA melting is created around the nic cleavage site, and the DNA can be cleaved by TrwC. For this second step, recognition of specific nucleotides is required to allow the formation of a U-shaped turn that locates the nic site at the catalytic center of TrwC. Finally, both the distal and proximal arms of IR2 are necessary for hairpin formation in the recipient cell. Thus, there are two distinguishable recognition sites, each for a different step of the processing reaction, both required for efficient conjugation. Because all the reported nic sites are located between 5 and 10 nucleotides from a more or less perfect inverted repeat (
      • Francia M.V.
      • Varsaki A.
      • Garcillán-Barcia M.P.
      • Latorre A.
      • Drainas C.
      • de la Cruz F.
      ), we propose that the above mechanism is a general mechanism shared by all of the conjugative relaxases. As a consequence, we hope our results and the two-step model in TrwC target recognition will have an application in the search and characterization of relaxase inhibitors that inhibit plasmid conjugation. In addition, they could help in the design of relaxase variants that can insert in specific genomic sequences, thus providing new tools for genomic engineering.

      REFERENCES

        • de la Cruz F.
        • Davies J.
        Trends Microbiol. 2000; 8: 128-133
        • Filutowicz M.
        • Burgess R.
        • Gamelli R.L.
        • Heinemann J.A.
        • Kurenbach B.
        • Rakowski S.A.
        • Shankar R.
        Plasmid. 2008; 60: 38-44
        • Potts R.G.
        • Lujan S.A.
        • Redinbo M.R.
        Future Microbiol. 2008; 3: 119-123
        • Fernandez-Lopez R.
        • Machón C.
        • Longshaw C.M.
        • Martin S.
        • Molin S.
        • Zechner E.L.
        • Espinosa M.
        • Lanka E.
        • de la Cruz F.
        Microbiology. 2005; 151: 3517-3526
        • Lujan S.A.
        • Guogas L.M.
        • Ragonese H.
        • Matson S.W.
        • Redinbo M.R.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 12282-12287
        • Llosa M.
        • de la Cruz F.
        Res. Microbiol. 2005; 156: 1-6
        • González-Pérez B.
        • Carballeira J.D.
        • Moncalián G.
        • de la Cruz F.
        Biotechnol. J. 2009; 4: 554-557
        • Zechner E.L.
        • de la Cruz F.
        • Eisenbrandt R.
        • Grahn A.M.
        • Koraimann G.
        • Lanka E.
        • Muth G.
        • Pansegrau W.
        • Thomas C.M.
        • Wilkins B.M.
        • Zatyka M.
        Thomas C.M. The Horizontal Gene Pool: Bacterial Plasmids and Gene Spread. Harwood Academic Publishers, Amsterdam2000
        • Gonzalez-Perez B.
        • Lucas M.
        • Cooke L.A.
        • Vyle J.S.
        • de la Cruz F.
        • Moncalián G.
        EMBO J. 2007; 26: 3847-3857
        • Garcillán-Barcia M.P.
        • Jurado P.
        • González-Pérez B.
        • Moncalián G.
        • Fernández L.A.
        • de la Cruz F.
        Mol. Microbiol. 2007; 63: 404-416
        • Grandoso G.
        • Llosa M.
        • Zabala J.C.
        • de la Cruz F.
        Eur. J. Biochem. 1994; 226: 403-412
        • Grandoso G.
        • Avila P.
        • Cayón A.
        • Hernando M.A.
        • Llosa M.
        • de la Cruz F.
        J. Mol. Biol. 2000; 295: 1163-1172
        • Llosa M.
        • Bolland S.
        • Grandoso G.
        • de la Cruz F.
        J. Bacteriol. 1994; 176: 3210-3217
        • Llosa M.
        • Grandoso G.
        • Hernando M.A.
        • de la Cruz F.
        J. Mol. Biol. 1996; 264: 56-67
        • Llosa M.
        • Grandoso G.
        • de la Cruz F.
        J. Mol. Biol. 1995; 246: 54-62
        • Reygers U.
        • Wessel R.
        • Müller H.
        • Hoffmann-Berling H.
        EMBO J. 1991; 10: 2689-2694
        • Matson S.W.
        Prog. Nucleic Acid Res. Mol. Biol. 1991; 40: 289-326
        • Fukuda H.
        • Ohtsubo E.
        J. Biol. Chem. 1995; 270: 21319-21325
        • Garcillán-Barcia M.P.
        • Francia M.V.
        • de la Cruz F.
        FEMS Microbiol. Rev. 2009; 33: 657-687
        • Francia M.V.
        • Varsaki A.
        • Garcillán-Barcia M.P.
        • Latorre A.
        • Drainas C.
        • de la Cruz F.
        FEMS Microbiol. Rev. 2004; 28: 79-100
        • Parker C.
        • Becker E.
        • Zhang X.
        • Jandle S.
        • Meyer R.
        Plasmid. 2005; 53: 113-118
        • Gao Q.
        • Luo Y.
        • Deonier R.C.
        Mol. Microbiol. 1994; 11: 449-458
        • Guasch A.
        • Lucas M.
        • Moncalián G.
        • Cabezas M.
        • Pérez-Luque R.
        • Gomis-Rüth F.X.
        • de la Cruz F.
        • Coll M.
        Nat. Struct. Biol. 2003; 10: 1002-1010
        • Grant S.G.
        • Jessee J.
        • Bloom F.R.
        • Hanahan D.
        Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 4645-4649
        • Jubete Y.
        • Maurizi M.R.
        • Gottesman S.
        J. Biol. Chem. 1996; 271: 30798-30803
        • Miroux B.
        • Walker J.E.
        J. Mol. Biol. 1996; 260: 289-298
        • Boer R.
        • Russi S.
        • Guasch A.
        • Lucas M.
        • Blanco A.G.
        • Pérez-Luque R.
        • Coll M.
        • de la Cruz F.
        J. Mol. Biol. 2006; 358: 857-869
        • Minton A.P.
        Schuster T.M. Laue T.M. Modern Analytical Ultracentrifugation. Birkhauser, Boston, MA1994
        • Laue T.M.
        • Shah B.D.
        • Ridgeway T.M.
        • Pelletier S.L.
        Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992
        • Philo J.S.
        Biophys. J. 1997; 72: 435-444
        • Schuck P.
        Biophys. J. 1998; 75: 1503-1512
        • Schuck P.
        • Rossmanith P.
        Biopolymers. 2000; 54: 328-341
        • Trask D.K.
        • DiDonato J.A.
        • Muller M.T.
        EMBO J. 1984; 3: 671-676
        • Llosa M.
        • Bolland S.
        • de la Cruz F.
        Mol. Gen. Genet. 1991; 226: 473-483
        • Martinez E.
        • de la Cruz F.
        Mol. Gen. Genet. 1988; 211: 320-325
        • Barabas O.
        • Ronning D.R.
        • Guynet C.
        • Hickman A.B.
        • Ton-Hoang B.
        • Chandler M.
        • Dyda F.
        Cell. 2008; 132: 208-220
        • Guynet C.
        • Hickman A.B.
        • Barabas O.
        • Dyda F.
        • Chandler M.
        • Ton-Hoang B.
        Mol. Cell. 2008; 29: 302-312
        • Becker E.C.
        • Meyer R.J.
        J. Mol. Biol. 2000; 300: 1067-1077
        • Datta S.
        • Larkin C.
        • Schildbach J.F.
        Structure. 2003; 11: 1369-1379
        • Hekman K.
        • Guja K.
        • Larkin C.
        • Schildbach J.F.
        Nucleic Acids Res. 2008; 36: 4565-4572
        • Williams S.L.
        • Schildbach J.F.
        Nucleic Acids Res. 2006; 34: 426-435
        • Rosenberg A.H.
        • Lade B.N.
        • Chui D.S.
        • Lin S.W.
        • Dunn J.J.
        • Studier F.W.
        Gene. 1987; 56: 125-135
        • Sarkar G.
        • Sommer S.S.
        BioTechniques. 1990; 8: 404-407