Conjugative transfer of plasmid DNA via close cell-cell junctions is the main route by which antibiotic resistance genes spread between bacterial strains. Relaxases are essential for conjugative transfer and act by cleaving DNA strands and forming covalent phosphotyrosine linkages. Based on data indicating that multityrosine relaxase enzymes can accommodate two phosphotyrosine intermediates within their divalent metal-containing active sites, we hypothesized that bisphosphonates would inhibit relaxase activity and conjugative DNA transfer. We identified bisphosphonates that are nanomolar inhibitors of the F plasmid conjugative relaxase in vitro. Furthermore, we used cell-based assays to demonstrate that these compounds are highly effective at preventing DNA transfer and at selectively killing cells harboring conjugative plasmids. Two potent inhibitors, clodronate and etidronate, are already clinically approved to treat bone loss. Thus, the inhibition of conjugative relaxases is a potentially novel antimicrobial approach, one that selectively targets bacteria capable of transferring antibiotic resistance and generating multidrug resistant strains.antimicrobial ͉ bacterial conjugation ͉ bisphosphonates ͉ F plasmid TraI ͉ relaxase inhibition C onjugative elements are responsible for the majority of horizontal gene transfers within and between bacterial strains (reviewed in ref. 1), as first described for the Escherichia coli F plasmid by Lederberg and Tatum in 1946 (2). Conjugative DNA transfer is also the central mechanism by which antibiotic resistance and virulence factors are propagated in bacterial populations (reviewed in ref.3). Indeed, it is well established that antibiotic resistance can be rapidly acquired in clinical settings and that such acquisition is critically dependent on conjugative DNA transfer (reviewed in ref. 4). Small-molecule inhibition of conjugation could prove to be a powerful method for curbing the generation and spread of multidrug-resistant strains. Past studies suggested that various antibiotics, polycyclic chemicals, and crude extracts inhibit conjugation at concentrations less than the antibacterial minimum inhibitory concentration (5-11); however, most of these effects have been attributed to nonconjugation-specific inhibition of bacterial growth or DNA synthesis (12)(13)(14)(15). This study describes a bottom-up approach used to identify the first small molecule inhibitors of conjugative DNA transfer that target an enzyme of the conjugative system.The DNA relaxase is a central enzyme in each conjugative system (16-18) and thus is a prime target for inhibition. The conjugative relaxase initiates DNA transfer with a site-and strand-specific ssDNA nick in the transferred strand (T-strand) at the origin of transfer (oriT), forming a covalent 5Ј-phosphotyrosine intermediate (16,(19)(20)(21)(22)(23). The nicked T-strand moves from the donor cell (plasmid ϩ ) to the recipient cell (plasmid Ϫ ) via an intercellular junction mediated by a type IV secretion system (reviewed in refs. 19, 24,...

The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site-and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5-to-3 helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first ϳ310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraI⌬252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraI⌬252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraI⌬252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.Conjugative DNA strand transfer is a highly conserved mechanism for unidirectional transfer of genetic information among bacteria of the same species, from one species to another and, in some instances, between kingdoms (for reviews, see references 15, 22, 25, 30, 37, and 44). Importantly, this is the mechanism used by most plasmids and conjugative transposons to facilitate their spread throughout a bacterial population, and the same underlying mechanism is used to transfer T-DNA from Agrobacterium tumefaciens to plant cells (23,45).The process of conjugative DNA transfer (CDT) is initiated with the formation of a stable mating pair between donor and recipient to establish the close cell-cell contact required for physical transfer of single-stranded DNA (ssDNA) from one cell to another. This is followed by the production of a site-and strand-specific nick at a locus called nic within the origin of transfer (oriT) or at the ends of T-DNA and the unwinding/ replication of the duplex DNA molecule to provide the ssDNA that enters the recipient. Within the recipient cell, host enzymes (primarily) convert the transferred ssDNA into doublestranded DNA that either circularizes to form a plasmid or is recombined into the recipient chromosome (for reviews, see references 15 and 44).Although reasonably well understood at this "macroscopic" level, the molecular details of the process by which DNA is transferred from donor to recipient are still being resolved. Key players are the proteins that initiate the physical transfer of ssDNA, the conjugative initiator proteins (7,22,30). These proteins introduce a site-and strand-specific ...

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3' Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5' Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.

SummaryConjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer -they introduce a site-and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5Ј-end of the cleaved DNA strand

TraI (DNA helicase I) is an Escherichia coli F plasmidencoded protein required for bacterial conjugative DNA transfer. The protein is a sequence-specific DNA transesterase that provides the site-and strand-specific nick required to initiate DNA strand transfer and a 5 to 3 DNA helicase that unwinds the F plasmid to provide the single-stranded DNA that is transferred from donor to recipient. Sequence comparisons with other transesterases and helicases suggest that these activities reside in the N-and C-terminal regions of TraI, respectively. Computer-assisted secondary structure probability analysis identified a potential interdomain region spanning residues 304 -309. Proteins encoded by segments of traI, whose N or C terminus either flanked or coincided with this region, were purified and assessed for catalytic activity. Amino acids 1-306 contain the transesterase activity, whereas amino acids 309 -1504 contain the helicase activity. The C-terminal 252 amino acids of the 1756-amino acid TraI protein are not required for either helicase or transesterase activity. Protein and nucleic acid sequence similarity searches indicate that the occurrence of both transesterase-and helicase-associated motifs in a conjugative DNA transfer initiator protein is rare. Only two examples (other than R100 plasmid TraI) were found: R388 plasmid TrwC and R46 plasmid (pKM101) TraH, belonging to the IncW and IncN groups of broad host range conjugative plasmids, respectively. The most significant structural difference between these proteins and TraI is that TraI contains an additional region of ϳ650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity.Bacterial conjugation is the primary mechanism by which many plasmids and conjugative transposons are spread throughout a bacterial population. The process begins with the formation of a stable mating pair involving a donor cell that contains a conjugative plasmid (or transposon) and a recipient cell that lacks the plasmid. This establishes the close cell-cell contact required for physical transfer of single-stranded DNA (ssDNA) 1 from the donor to the recipient. A site-and strandspecific nick is then introduced in oriT (origin of transfer), and the DNA is unwound to provide ssDNA for transfer to the recipient. Upon entering the recipient cell, the transferred ssDNA is converted into double-stranded DNA by host enzymes and either circularized to form a plasmid or recombined into the recipient chromosome. This stabilizes the transferred DNA in the recipient and ensures the transfer of genetic traits (for review, see Ref. 1).The enzymology of DNA strand transfer has been of interest since conjugation was first discovered over 50 years ago (2). In the last decade, it has become clear that transmissible plasmids encode a conjugative DNA transfer (CDT) initiator protein that plays a key role in initiating DNA strand transfer. These proteins nick their cognate supercoiled DNA substrate via a site-and strand-specific transes...