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

Abstract

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...

Host Range of the Conjugative Transfer System of IncP-9 Naphthalene-Catabolic Plasmid NAH7 and Characterization of Its oriT Region and Relaxase

“…3b) (19). One such IR in the R388 oriT region (IR 2 , which is the most proximal to the nic site) is a binding site of TrwC and essential for DNA cleavage at the nic site (20). The oriT N region carries four IRs in the fragment upstream of the nic site so that IR 4 (the most proximal IR to the nic site) is separated from it by 11 bases ( Fig.…”

ICEEc2, a New Integrative and Conjugative Element Belonging to the pKLC102/PAGI-2 Family, Identified inEscherichia coliStrain BEN374

“…We could not find within ICEEc2 a gene encoding a classical relaxase, an essential enzyme responsible for the formation of the relaxosome that replicates DNA prior to transfer (17). It is tempting to speculate that the two activities of these relaxases, namely, DNA nicking and DNA unwinding activities (30), are fulfilled by two independent proteins such as the ParB protein (ORF108) and the DnaB helicase (ORF109). Other genes that potentially contribute to the conjugation process include those that are related to plasmid replication, such as ssb, topB, and other DNA-related enzymes.…”

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

“…When the distal half of IR 2 is removed, TrwC binding affinity is decreased only 3-fold, and TrwC-mediated in vivo site-specific recombination is slightly affected (7). In contrast, the sequence 6ϩ2 (TTGTCT/AT) is absolutely required for TrwC binding, single-strand nicking, and strand transfer reactions (33). Similar results were reported for the related TraI relaxase of the F plasmid (46), where mutations affecting the nucleotides immediately adjacent to the nic site had a strong detrimental effect on conjugation.…”

“…The common 17-bp core sequence comprises a highly specific sequence termed the nic site, where the scissile phosphate lies, and a 5Ј region involving the recognition hairpin formed by an inverted repeat, IR 2 (17). This is the essential core sequence required in vivo for both conjugal mobilization (33) and site-specific recombination (7). When the distal half of IR 2 is removed, TrwC binding affinity is decreased only 3-fold, and TrwC-mediated in vivo site-specific recombination is slightly affected (7).…”