Changing the recognition site of a conjugative relaxase by rational design

González-Pérez, Blanca; Carballeira, José Daniel; Moncalián, Gabriel; Cruz, Fernando de la

doi:10.1002/biot.200800184

Cited by 13 publications

(16 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In the future, a bank of mutant relaxases could be obtained that target different sequences. In fact, the first such TrwC mutants have already been obtained (13). We are also conducting a mutagenesis analysis of TrwCrelated relaxases to obtain recombinase-proficient mutants that could be similarly used on other possible targets present in human and mammalian genomes.…”

Section: Discussionmentioning

confidence: 99%

Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences

Agúndez

Machón

César

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

TrwC is a bacterial protein involved in conjugative transfer of plasmid R388. It is transferred together with the DNA strand into the recipient bacterial cell, where it can integrate the conjugatively transferred DNA strand into its target sequence present in the recipient cell. Considering that bacterial conjugation can occur between bacteria and eukaryotic cells, this protein has great biotechnological potential as a site-specific integrase. We have searched for possible TrwC target sequences in the human genome. Recombination assays showed that TrwC efficiently catalyzes recombination between its natural target sequence and a discrete number of sequences, located in noncoding sites of the human genome, which resemble this target. We have determined the cellular localization of TrwC and derivatives in human cells by immunofluorescence and also by an indirect yeast-based assay to detect both nuclear import and export signals. The results indicate that the recombinase domain of TrwC (N600) has nuclear localization, but full-length TrwC locates in the cytoplasm, apparently due to the presence of a nuclear export signal in its C-terminal domain. The recombinase domain of TrwC can be transported to recipient cells by conjugation in the presence of the helicase domain of TrwC, but with very low efficiency. We mutagenized the trwC gene and selected for mutants with nuclear localization. We obtained one such mutant with a point A904T mutation and an extra peptide at its C terminus, which maintained its functionality in conjugation and recombination. This TrwC mutant could be useful for future TrwC-mediated site-specific integration assays in mammalian cells.

show abstract

Section: Discussionmentioning

confidence: 99%

Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences

Agúndez

Machón

César

et al. 2011

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…The chosen plasmid was pSU4298, which contains a C24G nic- site mutation (20) leading to a conjugation frequency of 10 −5 . These codon degeneracies and template were used to avoid the presence of the wild-type (wt) sequence in the final library.…”

Section: Methodsmentioning

confidence: 99%

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

Carballeira

González-Pérez

Moncalián

et al. 2014

Nucleic Acids Research

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…Moreover, bacterial relaxases might find a use as tools for site-specific DNA delivery to target eukaryotic cells for gene therapy (6). 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 (7).…”

mentioning

confidence: 99%

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

Lucas

González-Pérez

Cabezas

et al. 2010

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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 (IR 2 ). Mutation analysis in the nic region indicated that recognition of the IR 2 proximal arm and the nucleotides located between IR 2 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 IR 2 cruciform and recognition of the distal IR 2 arm and loop were not necessary for these reactions to take place. On the other hand, IR 2 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. IR 2 -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.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 (1). 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 (2, 3). Our group identified unsaturated fatty acids as conjugation inhibitors (4). 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. (5) 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 (6). 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 (7).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. 8 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 tyrosin...

show abstract

Changing the recognition site of a conjugative relaxase by rational design

Cited by 13 publications

References 15 publications

Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences

Nuclear Targeting of a Bacterial Integrase That Mediates Site-Specific Recombination between Bacterial and Human Target Sequences

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

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

Contact Info

Product

Resources

About