Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

Abstract

TraI, a bifunctional enzyme containing relaxase and helicase activities, initiates and drives the conjugative transfer of the Escherichia coli F plasmid. Here, we examined the structure and function of the TraI helicase. We show that TraI binds to singlestranded DNA (ssDNA) with a site size of ϳ25 nucleotides, which is significantly longer than the site size of other known superfamily I helicases. Low cooperativity was observed with the binding of TraI to ssDNA, and a double-stranded DNA-binding site was identified within the N-terminal region of TraI 1-858, outside the core helicase motifs of TraI. We have revealed that the affinity of TraI for DNA is negatively correlated with the ionic strength of the solution. The binding of AMPPNP or ADP results in a 3-fold increase in the affinity of TraI for ssDNA. Moreover, TraI prefers to bind ssDNA oligomers containing a single type of base. Finally, we elucidated the solution structure of TraI using small angle x-ray scattering. TraI exhibits an ellipsoidal shape in solution with four domains aligning along one axis. Taken together, these data result in the assembly of a model for the multidomain helicase activity of TraI.Conjugative plasmid transfer is a central mechanism for the horizontal exchange of genetic material between bacterial cells, as well as for the spread of antibiotic resistance genes and virulence factors (1-3). Conjugative plasmid transfer requires both a DNA relaxase and a DNA helicase. The relaxase initiates DNA transfer by cleaving the transferred strand at a specific site within the oriT, forming a 5Ј-phosphotyrosine intermediate. Following nicking, the helicase uses the energy from ATP hydrolysis to unwind the plasmid and drive the transfer of DNA into the recipient cell. The relaxase completes plasmid transfer by breaking the covalent phosphotyrosine linkage and releasing the transferred DNA for replication in the recipient (2, 4, 5).

Towards an integrated model of bacterial conjugation

“…Occasionally, an extra domain of unknown function is also observed. For instance, TraI, the relaxase of plasmid F, is a 182 kDa protein (Abdel-Monem, et al, 1983) with three distinct domains (Cheng, et al, 2011): the relaxase (1-306 aa), the helicase (309-1504) (Byrd, et al, 2002) and a third domain, comprising the last 252 aa, which is involved in DNA binding (Guogas, et al, 2009). In most cases, relaxases contain a conspicuous signature consisting of a histidine triad (3H motif) (Garcillan- Barcia, et al, 2009), which is used by the protein to bind divalent cations.…”

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

“…1) (1, 2, 25-27). Further inspection of the pCU1 sequence identified specific residues within these motifs associated with helicase activity, including an invariant lysine (Lys-507) in motif I/Walker A, the DExx box in motif II/Walker B (Asp-568 and Glu-569), and a conserved arginine (Arg-696) in motif IV, further suggesting that TraI was indeed an active helicase (1,2,24,(28)(29)(30).…”

“…Structures of the MOB F N-terminal relaxases from F TraI and R388 TrwC have been determined (40,41), and their enzymatic nicking activity has been thoroughly investigated (42)(43)(44)(45)(46)(47). Although DNA binding of the F TraI C-terminal helicase has recently been investigated (30,48), much less is known about the structure and mechanism of these C-terminal helicase domains relative to the N-terminal relaxase domains. Studies of F TraI and R388 TrwC demonstrate, however, that both enzymatic activities of the relaxase and helicase are required for CPT (24,38,44).…”

“…Conjugative plasmid transfer (CPT) mediates the propagation of antibiotic resistance genes and virulence factors within commensal and pathogenic bacteria (25-27) and relies on the helicase domain of a bifunctional protein to facilitate plasmid DNA transfer (24,(28)(29)(30). Early work characterizing the general mechanism of CPT was accomplished through the study of the F (fertility) plasmid system, a member of the MOB F family of conjugative plasmids (31-35).…”

“…Despite their importance, SF1B helicases are still poorly characterized in comparison to SF1A and SF2 helicases, though some recent studies have expanded our knowledge (19-21). SF1B helicases are critical to a diverse range of processes, including DNA repair by human DNA helicase B (22), telomere regulation by Pif1 (23), and conjugative plasmid transfer by F TraI (24).Conjugative plasmid transfer (CPT) mediates the propagation of antibiotic resistance genes and virulence factors within commensal and pathogenic bacteria (25-27) and relies on the helicase domain of a bifunctional protein to facilitate plasmid DNA transfer (24,(28)(29)(30). Early work characterizing the general mechanism of CPT was accomplished through the study of the F (fertility) plasmid system, a member of the MOB F family of conjugative plasmids (31-35).…”