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• W2019941447 abstract "Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21–32 yielded a mutant protein, TrfA-44Δ2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37–55 (TrfA-44Δ3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71–88 (TrfA-44Δ4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication. Broad host range plasmid RK2 encodes two versions of its essential replication initiation protein, TrfA, using in-frame translational starts spaced 97 amino acids apart. The smaller protein, TrfA-33, is sufficient for plasmid replication in many bacterial hosts. Efficient replication in Pseudomonas aeruginosa, however, specifically requires the larger TrfA-44 protein. With the aim of identifying sequences of TrfA-44 required for stable replication of RK2 in P. aeruginosa, specific deletions and a substitution mutant within the N terminus sequence unique to TrfA-44 were constructed, and the mutant proteins were tested for activity. Deletion mutants were targeted to three of the four predicted helical regions in the first 97 amino acids of TrfA-44. Deletion of TrfA-44 amino acids 21–32 yielded a mutant protein, TrfA-44Δ2, that had lost the ability to bind and load the DnaB helicase of P. aeruginosa or Pseudomonas putida onto the RK2 origin in vitro and did not support stable replication of an RK2 mini-replicon in P. aeruginosa in vivo. A substitution of amino acid 22 within this essential region resulted in a protein, TrfA-44E22A, with reduced activity in vitro, particularly with the P. putida helicase. Deletion of amino acids 37–55 (TrfA-44Δ3) slightly affected protein activity in vitro with the P. aeruginosa helicase and significantly with the P. putida helicase, whereas deletion of amino acids 71–88 (TrfA-44Δ4) had no effect on TrfA activity in vitro with either helicase. These results identify regions of the TrfA-44 protein that are required for recruitment of the Pseudomonas DnaB helicases in the initiation of RK2 replication. A critical step in the initiation of DNA replication is the recruitment, loading, and activation of the replicative helicase. Helicase activity is not only necessary for progression of the replication fork but, as studies with the Escherichia coli chromosomal origin oriC have revealed, the DnaB helicase makes essential contacts with other proteins in the replication complex. Recruitment of the helicase in E. coli requires association of the DnaB hexamer with the E. coli DnaC accessory protein (1Funnell B. Baker T. Kornberg A. J. Biol. Chem. 1987; 262: 10327-10334Abstract Full Text PDF PubMed Google Scholar, 2Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 3Kobori J. Kornberg A. J. Biol. Chem. 1982; 257: 13770-13775Abstract Full Text PDF PubMed Google Scholar). The DnaB-DnaC complex interacts with the host initiation protein, DnaA, bound to specific sequences (DnaA boxes) at oriC. This association results in the loading of the helicase onto unwound single-stranded DNA in the origin and the release of DnaC (4Messer W. Blaesing F. Majka J. Nardmann J. Schaper S. Schmidt A. Seitz H. Speck C. Tuengler D. Wegrzyn G. Weigel C. Welzeck M. Zakrzewska-Czerwinska J. Biochimie (Paris). 1999; 81: 819-825Crossref PubMed Scopus (79) Google Scholar, 5Marszalek J. Kaguni J.M. J. Biol. Chem. 1994; 269: 4883-4890Abstract Full Text PDF PubMed Google Scholar, 6Sutton M.D. Carr K.M. Vicente M. Kaguni J.M. J. Biol. Chem. 1998; 273: 34255-34262Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 7Marszalek J. Zhang W. Hupp T.R. Margulies C. Carr K.M. Cherry S. Kaguni J.M. J. Biol. Chem. 1996; 271: 18535-18542Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Once loaded onto the single-stranded DNA, DnaB interacts directly with DnaG to facilitate primase loading onto the single-stranded DNA at the replication fork (8Tougu K. Marians K.J. J. Biol. Chem. 1996; 271: 21391-21397Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Lu Y.-B. Ratnakar P.V.A.L. Mohanty B.K. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12902-12907Crossref PubMed Scopus (99) Google Scholar). This interaction becomes the primary regulator of Okazaki fragment synthesis (10Tougu K. Marians K.J. J. Biol. Chem. 1996; 271: 21398-21405Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) and also ensures the proper placement of primers for leading strand synthesis (11Hiasa H. Marians K.J. J. Biol. Chem. 1999; 274: 27244-27248Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The subsequent association of DnaB with the Tau subunit of the DNA polymerase III holoenzyme results in rapid movement of the replication fork (12Kim S. Dallmann H.G. McHenry C.S. Marians K.J. Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar, 13Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar).Studies on plasmids and phages that replicate in E. coli have revealed additional strategies for recruiting DnaB to a replication origin. The replication initiation proteins encoded by the narrow host range plasmids pSC101 and R6K, RepA and π, respectively, have been shown to interact with DnaB in vitro (14Datta H.J. Khatri G.S. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 73-78Crossref PubMed Scopus (54) Google Scholar, 15Lu Y.-B. Datta H.J. Bastia D. EMBO J. 1998; 17: 5192-5200Crossref PubMed Scopus (63) Google Scholar, 16Ratnakar P.V. Mohanty B.K. Lobert M. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5522-5526Crossref PubMed Scopus (48) Google Scholar). For plasmid pSC101, the RepA-DnaB interaction is essential for loading the helicase onto the origin in a process that also requires the DnaC and DnaA proteins (14Datta H.J. Khatri G.S. Bastia D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 73-78Crossref PubMed Scopus (54) Google Scholar). Bacteriophage λ utilizes the phage-encoded P protein instead of the host-encoded DnaC protein to recruit DnaB to the initiation complex (17Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10709-10718Abstract Full Text PDF PubMed Google Scholar, 18Alfano C. McMacken R. J. Biol. Chem. 1989; 264: 10699-10708Abstract Full Text PDF PubMed Google Scholar). With phage P2, the phage-encoded B protein appears to be required for DnaB recruitment for lytic replication, although not replication of P2 as a plasmid (19Odegrip R. Schoen S. Haggard-Ljungquist E. Park K. Chattoraj D.K. J. Virol. 2000; 74: 4057-4063Crossref PubMed Scopus (20) Google Scholar).Broad host range plasmids are able to replicate in diverse bacteria. As such, they provide the means to examine replication of a specific replicon in different host backgrounds. RK2 is a promiscuous plasmid belonging to the IncP group. It is noted for its ability to transfer into and be stably maintained in a wide variety of Gram-negative bacteria. Only two regions of an IncP plasmid are essential for broad host range replication, the cis acting origin for DNA replication (oriV) and the trfA gene that encodes two forms of a trans-acting replication initiation protein. The host bacterium provides all other proteins essential for replication. The simplicity of this system has allowed for a direct comparison of the mechanism for DNA replication initiation in different bacterial species (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar, 21Caspi R. Helinski D.R. Pacek M. Konieczny I. J. Biol. Chem. 2000; 275: 18454-18461Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar).IncP plasmids can be divided into two subgroups based on sequence differences: IncPα, to which RK2 belongs, and IncPβ, which is represented by the plasmid R751. Both RK2 and R751 can replicate in E. coli and Pseudomonas aeruginosa. Earlier studies had shown that the smaller form of the RK2 initiation protein, TrfA-33, was sufficient for stable replication of an RK2 mini-replicon in E. coli or Pseudomonas putida but that the larger form of the protein, TrfA-44, was required for plasmid replication in P. aeruginosa (22Shingler V. Thomas C.M. Biochim. Biophys. Acta. 1989; 1007: 301-308Crossref PubMed Scopus (16) Google Scholar, 23Fang F.C. Helinski D.R. J. Bacteriol. 1991; 173: 5861-5868Crossref PubMed Google Scholar, 24Durland R.H. Helinski D.R. Plasmid. 1987; 18: 164-169Crossref PubMed Scopus (57) Google Scholar). Recently, it has been shown that TrfA-44 is unique among plasmid initiation proteins in that it can load and activate the DnaB helicase of P. aeruginosa or P. putida on the RK2 origin in vitro in the absence of the DnaA protein (25Jiang Y. Pacek M. Helinski D.R. Konieczny I. Toukdarian A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8692-8697Crossref PubMed Scopus (29) Google Scholar). By contrast, the TrfA-33 protein requires DnaA protein to load and activate the helicase of P. putida and requires DnaA plus DnaC to load the helicase of E. coli. Consistent with the earlier in vivo studies, TrfA-33 did not function in vitro with the DnaB helicase of P. aeruginosa either in the presence or in the absence of P. aeruginosa DnaA (25Jiang Y. Pacek M. Helinski D.R. Konieczny I. Toukdarian A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8692-8697Crossref PubMed Scopus (29) Google Scholar).Since it has been shown that the open complex formed on a supercoiled oriV template is indistinguishable when either TrfA-33 or TrfA-44 proteins are used with the DnaA proteins of E. coli, P. putida, or P. aeruginosa (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar), the observed differences in activity of the two forms of the TrfA initiation protein in P. aeruginosa are likely due to a difference in their ability to interact with the P. aeruginosa DnaB helicase. This suggested a role for the N-terminal 97 amino acids, which are unique to TrfA-44, in DnaB recruitment in Pseudomonas. We therefore undertook a mutational analysis of the first 97 amino acids of this protein in an attempt to identify those regions within the N terminus that are important in the loading of the Pseudomonas helicases at the RK2 replication origin.EXPERIMENTAL PROCEDURESConstruction of trfA Mutations—Plasmid pGC1 (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar) was used to express His6TrfA-44(M98L/G254D/S267L), a variant of TrfA-44 that has six histidine residues inserted between the first amino acid (Met) and the second amino acid (Asn) of the native protein to allow for ease of purification, the M98L amino acid substitution that replaces the native methionine start of TrfA-33 with a leucine and thus eliminates TrfA-33 expression, and the G254D and S267L changes that result in a primarily monomeric form of the protein. A previous study had shown that although wild type TrfA protein is primarily a dimer in solution, it was the monomeric form that was essential for replication initiation activity (26Toukdarian A.E. Helinski D.R. Perri S. J. Biol. Chem. 1996; 271: 7072-7078Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Plasmid pGC1 served as the template for the construction of three specific N-terminal deletions and one point mutant as follows.Amino acids 21–32, 37–55, or 71–88 (numbering based on the native non-His-tagged protein) were deleted by using a PCR-based strategy. For each deletion, two pairs of primers were used to synthesize fragments that flanked either side of the desired deletion in the trfA gene. Both PCR fragments included restriction enzyme sites such that after digestion with the appropriate enzymes, ligation of the two fragments in the presence of a vector backbone resulted in the construction of a pGC1 derivative. These derivatives expressed the desired TrfA-44 deletion protein, which contained either one or two additional amino acids at the site of the deletion. The resulting plasmids were pZZ28, for expression of TrfA-44Δ2, pZZ25, for expression of TrfA-44Δ3, and pZZ23, for expression of TrfA-44Δ4.To change amino acid residue 22 in the trfA gene of pGC1 from Glu (GAG) to Ala (GCG), and coincidentally introduce a SacII site, primer 5′-GGGTTTTCCGCCGCGGATGCCGAAAC-3′ and its complement were used with pGC1 template and the PCR-based QuikChange site-directed mutagenesis kit (Stratagene) to introduce the underlined base change. The EcoRI-SfiI fragment of one resulting mutant was sequenced to confirm the presence of the desired mutation, and this fragment was then used to replace the matching fragment in non-mutagenized pGC1 resulting in pZZ29.Source of Proteins—The N-terminal His6-tagged G254D/S267L form of TrfA-33 (27Blasina A. Kittell B.L. Toukdarian A.E. Helinski D.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3559-3564Crossref PubMed Scopus (56) Google Scholar), TrfA-44 (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar), and the TrfA-44 N-terminal point and deletion mutants were used throughout this study. Plasmids expressing the TrfA-44 N-terminal mutants were transferred into E. coli strain JP313 to overexpress and purify the proteins as described previously for TrfA-44 (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar).C-terminal His6-tagged DnaA proteins of E. coli, P. putida, and P. aeruginosa (21Caspi R. Helinski D.R. Pacek M. Konieczny I. J. Biol. Chem. 2000; 275: 18454-18461Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and C-terminal His6-tagged DnaB proteins of E. coli, P. putida, and P. aeruginosa (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar) were purified as described. The modified proteins have been found to behave similarly to the native E. coli proteins in several in vitro assays (28Konieczny I. Doran K.S. Helinski D.R. Blasina A. J. Biol. Chem. 1997; 272: 20173-20178Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 29Konieczny I. Helinski D.R. J. Biol. Chem. 1997; 272: 33312-33318Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). E. coli DnaC and E. coli DNA gyrase were purified from strains RSC680 and AN1459(pPS562), respectively, kindly provided along with purification protocols by Dr. Nick Dixon. HU was a generous gift from Dr. Roger McMacken. Commercially available proteins were SSB 1The abbreviation used is: SSB, single-stranded DNA binding protein. (Promega), creatine kinase and bovine serum albumin (Fraction V) (Sigma), and DNA restriction and modification enzymes from various commercial sources.Helicase Unwinding Assay—Helicase unwinding assays (FI* formation) were performed as described previously (29Konieczny I. Helinski D.R. J. Biol. Chem. 1997; 272: 33312-33318Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) using 300 ng of the mini-RK2 replicon pKD19L1 (30Doran K.S. Konieczny I. Helinski D.R. J. Biol. Chem. 1998; 273: 8447-8453Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) as the DNA template. The standard assay contained: DnaA (as noted), DnaB (as noted), DnaC (200 ng), TrfA-33 or TrfA-44 (300 ng), HU (2 ng), gyrase (120 ng), and SSB (230 ng).Gel Exclusion Chromatography—Reaction mixes containing 1200 ng of the RK2 mini-replicon pTJS42 (31Schmidhauser T.J. Filutowicz M. Helinski D.R. Plasmid. 1983; 9: 325-330Crossref PubMed Scopus (52) Google Scholar), 2400 ng of P. aeruginosa DnaB, and 3200 ng of TrfA-33, TrfA-44, or TrfA-44 mutants were incubated at 33 °C for 30 min as described previously (29Konieczny I. Helinski D.R. J. Biol. Chem. 1997; 272: 33312-33318Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The reactions were run on 1.75-ml columns of Sepharose CL-4B. Fractions were collected and analyzed on SDS-PAGE followed by Western blotting using a mouse antibody specific to the His tag (Tetra-His antibody, Qiagen). Bound primary antibody was detected using horseradish peroxidase-conjugated goat anti-mouse IgG followed by SuperSignal West Pico (Pierce). TrfA protein was detected separately by applying aliquots of each fraction onto a nitrocellulose membrane using a slot-blot device followed by incubation with rabbit anti-TrfA antibody. Bound rabbit antibody was detected using horseradish peroxidase-conjugated goat anti-rabbit IgG followed by SuperSignal West Pico (Pierce).Plasmid Stability in Vivo—To test the ability of the TrfA-44 mutants to support replication in P. aeruginosa, the mutations were transferred into the RK2 mini-replicon pRR10 (32Roberts R.C. Burioni R. Helinski D.R. J. Bacteriol. 1990; 172: 6204-6216Crossref PubMed Google Scholar). The EcoRI-SfiI fragment containing the 5′ end of trfA of plasmid pRR10 was replaced with the EcoRI-SfiI fragment containing the 5′ end of trfA from plasmids pZZ23 (TrfA-44Δ4), pZZ25 (TrfA-44Δ3), pZZ28 (TrfA-44Δ2), pZZ29 (TrfA-44E22A), and pGC1 (TrfA-44), resulting in plasmids pRR10-98HisΔ4, pRR10-98HisΔ3, pRR10-98HisΔ2, pRR10-98HisE22A, and pRR10-98His, respectively. E. coli strain MC1029 Ch3/86, which expresses wild type TrfA-33 and TrfA-44 proteins, was used as the host for the construction (33Thomas C.M. Stalker D.M. Helinski D.R. Mol. Gen. Genet. 1981; 181: 1-7Crossref PubMed Scopus (53) Google Scholar).The five pRR10 derivatives were then introduced separately into P. aeruginosa by electroporation (34Dennis J.J. Sokol P.A. Nickoloff J.A. Electroporation Protocols for Microorganisms. Vol. 47. Humana Press, Totowa, NJ1995: 125-133Google Scholar) using 100 μg/ml carbenicillin for selection. A single colony for each of these five strains was picked from a fresh overnight LB + 100 μg/ml carbenicillin plate, resuspended in 5 ml of LB, and then incubated for 24 h with shaking at 37 °C. At subsequent time points, 50 μl of the stationary phase culture was transferred to 5 ml of LB broth, and incubation at 37 °C with shaking was continued. In addition, serial dilutions of the initial suspension and the subsequent overnight cultures were prepared, and aliquots were plated onto LB agar without antibiotics. After overnight incubation at 37 °C, 100 colonies from these plates were patched to plates with LB + 100 μg/ml carbenicillin and then onto LB plates to determine the percentage of cells still maintaining the plasmid.RESULTSRationale for the Construction of Specific Mutants—Plasmids RK2 (IncPα replicon) and R751 (IncPβ replicon) can replicate in E. coli and P. aeruginosa, and both plasmids express two forms of the essential plasmid-encoded replication initiation protein. The larger form of the RK2 initiation protein, TrfA-44, is required for replication of the plasmid in P. aeruginosa, whereas the smaller form is sufficient for replication in E. coli (22Shingler V. Thomas C.M. Biochim. Biophys. Acta. 1989; 1007: 301-308Crossref PubMed Scopus (16) Google Scholar, 23Fang F.C. Helinski D.R. J. Bacteriol. 1991; 173: 5861-5868Crossref PubMed Google Scholar, 24Durland R.H. Helinski D.R. Plasmid. 1987; 18: 164-169Crossref PubMed Scopus (57) Google Scholar). The smaller forms of the RK2 and the R751 initiation proteins, TrfA-33 and TrfA2, respectively, are each 285 amino acids in length, and when aligned using ClustalW (35Thompson J. Higgins D. Gibson T. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55151) Google Scholar), 91% of the residues are either identical or strongly similar (data not shown). The larger forms of the initiation protein of the two plasmids differ in size and are less related in the N-terminal regions. Alignment of the 382-amino-acid TrfA-44 protein of RK2 and the 407-amino-acid TrfA1 protein of R751 using ClustalW identified 78% residues that were identical or strongly similar. The alignment result for the N terminus of the larger proteins is shown in Fig. 1. Given the increased sequence variation of the two larger proteins as compared with the two smaller proteins, it seemed likely that those residues conserved in the N terminus of TrfA-44 and TrfA1 would be important for protein activity in P. aeruginosa.Analysis of TrfA-44 using the secondary structure prediction program PredictProtein (36Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar) identified four possible helical regions in the first 97 amino acids of the protein (Fig. 1). The fourth helical region overlapped with a region of striking similarity between TrfA-44 and TrfA1, comprised of amino acid residues 70–90 of TrfA-44, that had been noted in an earlier study (37Thorsted P.B. Shah D.S. Macartney D. Kostelidou K. Thomas C.M. Plasmid. 1996; 36: 95-111Crossref PubMed Scopus (26) Google Scholar). We therefore decided to delete amino acids 71–88 of TrfA-44, which encompassed this helical region. The analysis of the in vitro activity of this deletion mutant, designated as TrfA-44Δ4, led to the construction of deletions TrfA-44Δ3 (amino acid residues 37–55 inclusive deleted) and TrfA-44Δ2 (amino acid residues 21–32 inclusive deleted), which removed, separately, two of the three remaining predicted helical regions.In Vitro Helicase Loading Activity of TrfA-44 Deletion Mutants—Loading and activation of DnaB helicase at a replication origin on a supercoiled plasmid template can be examined in vitro using the FI* assay (2Baker T.A. Sekimizu K. Funnell B.E. Kornberg A. Cell. 1986; 45: 53-64Abstract Full Text PDF PubMed Scopus (135) Google Scholar, 29Konieczny I. Helinski D.R. J. Biol. Chem. 1997; 272: 33312-33318Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The basis of this assay is that helicase unwinding of a supercoiled template in the presence of DNA gyrase and SSB produces a highly unwound form of the DNA, termed FI*, that can be distinguished electrophoretically from the template FI form.The FI* assay was used to test the functionality of the mutant TrfA proteins in the recruitment, loading, and activation of DnaB at the RK2 origin. To ensure that slight differences in the activity of the mutants would be seen, the amount of DnaB helicase added to the standard assay was titrated to determine the lowest level of DnaB protein needed to get full activity with wild type TrfA-44 protein. The level of E. coli DnaB necessary for full activity, 1600 ng, was similar to that reported previously (29Konieczny I. Helinski D.R. J. Biol. Chem. 1997; 272: 33312-33318Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and the addition of both E. coli DnaA and DnaC proteins was essential (data not shown). The level of Pseudomonas DnaB required for full activity with wild type TrfA-44 protein, 50 ng for DnaB from P. aeruginosa and 100 ng for P. putida DnaB (Fig. 2), was less then reported previously (20Caspi R. Pacek M. Consiglieri G. Helinski D.R. Toukdarian A. Konieczny I. EMBO J. 2001; 20: 3262-3271Crossref PubMed Scopus (54) Google Scholar). As expected from previous work (25Jiang Y. Pacek M. Helinski D.R. Konieczny I. Toukdarian A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 8692-8697Crossref PubMed Scopus (29) Google Scholar), activity with TrfA-44 was not dependent on the addition of Pseudomonas DnaA (compare Fig. 2, lane 5 or 6, with lane 4, and compare lane 11 or 12 with lane 10). We then confirmed that TrfA-33 was able to load this lower level of P. putida helicase, but only in the presence of P. putida DnaA (Fig. 2, lanes 7–9), and that even with DnaA present, P. aeruginosa DnaB, at this lower concentration, could not be loaded by TrfA-33 (Fig. 2, lanes 2 and 3).Fig. 2Activity of P. aeruginosa and P. putida DnaB helicase at the RK2 origin as measured by the FI* assay. Assays were performed in the presence of 50 ng of P. aeruginosa DnaB (lanes 1–6)or 100 ng of P. putida DnaB (lanes 7–12), the homologous DnaA protein (0, 200, or 600 ng indicated as –, +, or ++), and 300 ng of TrfA-33 or TrfA-44 as indicated. The positions of the FI* (covalently closed highly underwound), FI (covalently closed supercoiled), FII (open circular), and FIII (linear) forms of the template DNA are noted by arrows.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The ability of the three TrfA-44 deletion mutant proteins to load P. aeruginosa or P. putida helicase onto RK2 oriV was then tested. As shown in Fig. 3, TrfA-44Δ4 (lanes 5) was fully functional with DnaB from P. aeruginosa (A) or P. putida (B). TrfA-44Δ3 (lanes 4) had reduced activity, particularly with P. putida DnaB, whereas TrfA-44Δ2 (lanes 3) was not functional with either helicase. Activity with TrfA-44Δ2 was not restored by the addition of P. aeruginosa DnaA to reactions containing P. aeruginosa DnaB but was restored by the addition of P. putida DnaA to reactions containing P. putida DnaB protein (data not shown).Fig. 3Ability of TrfA-44 N-terminal deletion mutants to load and activate the Pseudomonas DnaB helicase in the absence of DnaA. FI* assays were performed with P. aeruginosa DnaB (50 ng) (A) or P. putida DnaB (100 ng) (B) and 300 ng of TrfA-44wt (lanes 2), TrfA-44Δ2 (lanes 3), TrfA-44Δ3 (lanes 4), TrfA-44Δ4 (lanes 5), or no TrfA protein (lanes 1). wt, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All three mutant proteins functioned as well as the wild type protein in the loading and activation of E. coli DnaB in the presence of E. coli DnaA and DnaC (Fig. 4). These results showed that the observed defects in the TrfA-44Δ2 and TrfA-44Δ3 mutants were specific to the recruitment and loading of DnaB from Pseudomonas in the absence of DnaA.Fig. 4TrfA-44 mutants are fully functional in loading the E. coli DnaB helicase in the presence of the E. coli DnaA and DnaC proteins. FI* assays were performed in the presence of E. coli DnaA (80 ng), DnaB (1600 ng), and DnaC (200 ng) with 300 ng of wild type (wt) or mutant TrfA-44 proteins as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Point Mutation in TrfA-44 with Altered Helicase Loading Activity—The TrfA-44Δ2 mutant protein was deleted for 12 amino acids. To determine whether a specific amino acid within the deleted region was required for helicase loading, we constructed a specific point mutant. Reasoning that a charged and/or polar amino acid residue might be involved in the stabilization of a DnaB/TrfA-44 interaction, we replaced the glutamate residue at position 22 with alanine, yielding TrfA-44E22A. This change alters the charge and polarity at this position but not the helical nature of this region as predicted using PredictProtein.The TrfA-44E22A mutant protein was tested for helicase loading activity. This mutant protein, when added at 300 ng/assay, had slightly reduced activity when compared with wild type TrfA-44 protein with P. aeruginosa or P. putida DnaB (data not shown). This reduction in activity was more pronounced, particularly with P. putida DnaB, when the amount of TrfA protein added to each reaction was decreased to 200 ng/assay (Fig. 5), suggesting that the point mutant was also altered in helicase recruitment or loading. The TrfA-44E22A mutant protein behaved as the wild type protein at all concentrations tested in assays with E. coli DnaA, DnaB, and DnaC (data not shown).Fig. 5The effect of a point mutant of TrfA-44 on the loading and activation of Pseudomonas DnaB helicase at the RK2 replication origin. FI* assays were performed in the presence of 50 ng of P. aeruginosa DnaB (lanes 1–3) or 100 ng of P. putida DnaB (lanes 4–6) and 200 ng of TrfA-44wt (lanes 1 and 4), TrfA-44Δ2 (lanes 2 and 5), or TrfA-44E22A point mutant (lanes 3 and 6) proteins. wt, wild type.View Large Image Figure ViewerDownload Hi-res i" @default.
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• W2019941447 title "A Specific Region in the N Terminus of a Replication Initiation Protein of Plasmid RK2 Is Required for Recruitment of Pseudomonas aeruginosa DnaB Helicase to the Plasmid Origin" @default.
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