A high security double lock and key mechanism in HUH relaxases controls oriT-processing for plasmid conjugation

Abstract

Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC-DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome.

Figure 1.

Models of conjugative DNA processing by Y1 or Y2 relaxases (modified from (7) and (8)). I, conjugation is initiated by the relaxase recognizing the proximal arm of an inverted repeat (IR₂) adjacent to the nic-site. II, relaxase binding allows the formation of a single-stranded DNA (ssDNA) U-turn that positions the nic-site at the relaxase active site. The U-turn is stabilized by the ‘fingers’ subdomain of Y2 relaxases. After nic-cleavage, a covalent phosphotyrosine bond between the cleaved DNA strand and the relaxase is formed. III, subsequent DNA strand displacement generates the DNA single strand that, piloted by the relaxase, is transferred into the recipient cell. IV, In Y2 relaxases, a second tyrosine present in the same molecule attacks the newly formed nic-site to generate a free 3′OH end able to recircularize the transferred plasmid DNA. In Y1 relaxases, either the free 3′OH is released in the donor cell (monomeric Y1 model) or a second relaxase molecule provides the free tyrosine that attacks the newly formed nic-site (dimeric Y1 model).

Figure 2.

Experimental outline depicting the selection system for the study of TrwC specificity. Library construction: Quickchange allows the construction of a library of mutants at the nic-site. DH5a cells were transformed with the plasmid library by electroporation. Conjugation: mobilizable plasmids were selected by mating library-containing DH5a donor cells with recipient UB1637 cells. Pooled donor colonies as well as pooled transconjugant colonies were sequenced to be compared. See ‘Materials and Methods’ section for details.

Figure 3.

Full randomization libraries. Electropherogram showing the randomized region in the transconjugant pool sequence of each library tested (A–H). Essential nucleotide positions are colored in red, positions that admit only some mutations in orange and positions where any nucleotide can be located appear in green.

Figure 4.

Classification of different specificity regions within the nic sequence according to the results obtained from randomization libraries. 2D representation of the nucleoprotein complex according to PDB ID: 1QX0 (19). Hydrogen bonds are indicated with green lines and van der Waals contacts with gray dashed lines. Nucleotides are colored like in Figure 3 according to the results obtained in the full randomization libraries plus those ones obtained from the partially randomized libraries of the ssDNA region. Nucleotides involved in the U-turn stabilization are colored in blue.

Figure 5.

Representation of the U-shaped turns in the structures of the nucleoprotein complexes R388_TrwC_R:mutant IV, R388_TrwC_R:nic-site (PDB ID: 2CDM,(23));F_TraI:nic-site (PDB ID: 2A0I (31)) and pLW1043_NES: nic-site (PDB ID: 4HT4 (32). The nucleotides are numbered equivalent to R388_TrwC structure. The position of the nic-cleavage site is highlighted by an orange arrow. Hydrogen bonds are represented by dashed lines. The alignment of representative nic-sites of MOB_F and MOB_Q plasmids is shown. U-shaped turn forming nucleotides are shown in bold while nucleotides changed in mutant IV are shown in red.

Figure 6.

(A) Supercoiled DNA cleavage of wild-type nic sequence and nic mutant IV by TrwC. Reactions were carried out as described in ‘Material and Methods’ section and products resolved in a 0.8% agarose gel. Growing concentrations of purified TrwC_R were added to reaction mixtures containing pSU4910 (wild-type) or mutant IV SC DNA (200 ng): lane L, DNA ladder; lanes 1 and 5, no TrwC_R added, lanes 2 and 6, TrwC_R 0.3 μM; lanes 3 and 7, TrwC_R 3 μM; lanes 4 and 8, TrwC_R 30 μM;. The position of nicked DNA and SC DNA is indicated. (B) Single stranded oligonucleotide cleavage assays followed by capillary electrophoresis. Growing concentrations of purified TrwC_R were added to reaction mixtures containing 50 nm 5′-fluorescein-labeled oligonucleotides R(25 + 18) (wild-type) or RIV(25 + 18) (Mutant IV). (1) no TrwC_R added; (2) TrwC_R 0.3 μM; (3) TrwC_R 3 μM; (4) TrwC_R 30 μM. The positions of the cleaved oligonucleotides are shown by asterisks.