Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.     

Purified Escherichia coli F-factor TraY protein binds oriT.

The traY gene of the Escherichia coli F plasmid has been shown by genetic studies (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980) to be involved in the site-specific nicking reaction at oriT required for the initiation of DNA transfer during bacterial conjugation. In order to assign a biochemical function to TraY protein, the traY gene was cloned in a plasmid vector which utilizes the strong T7 phi 10 promoter to overproduce the protein. The plasmid-encoded TraY protein was specifically labeled with [35S]methionine, and purification of the polypeptide was accomplished by monitoring the radioactive label. Purified TraY protein had a relative molecular mass of approximately 17,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino terminus of the purified protein was sequenced to confirm that the protein was encoded by the traY gene. The protein sequence revealed that the start codon for the TraY protein was a UUG codon 36 base pairs upstream of the AUG start site originally deduced from the DNA sequence (T. Fowler, L. Taylor, and R. Thompson, Gene 26:79-89, 1983). This start sequence confirmed the premise of Inamoto et al. that the F-plasmid TraY polypeptide-coding sequence would begin with UUG, creating a reading frame which renders a large degree of amino acid sequence identity with the TraY polypeptide from R100 (S. Inamoto, Y. Yoshioka, and E. Ohtsubo, J. Bacteriol. 170:2749-2757, 1988). The purified TraY protein from F bound specifically to the origin of transfer region of the F plasmid. However, no nicking activity was detected at oriT by using TraY protein or TraY protein in conjunction with helicase I.     

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.     

Y-box-binding protein 1 stimulates abasic site cleavage

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5′ side of the AP site, thereby generating a single-strand DNA break flanked by the 3′-OH and 5′-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations.     

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.     

Specific DNA binding of the TraM protein to the oriT region of plasmid R100.

The product of the traM gene of plasmid R100 was purified as the TraM-collagen-beta-galactosidase fusion protein (TraM*) by using a beta-galactosidase-specific affinity column, and the TraM portion of TraM* (TraM') was separated by collagenolysis. Both the TraM* and TraM' proteins were found to bind specifically to a broad region preceding the traM gene. This region (designated sbm) was located within the nonconserved region in oriT among conjugative plasmids related to R100. The region seems to contain four core binding sites (designated sbmA, sbmB, sbmC, and sbmD), each consisting of a similar number of nucleotides and including a homologous 15-bp sequence. This result, together with the observation that the TraM* protein was located in the membrane fraction, indicates the possibility that the TraM protein has a function in anchoring the oriT region of R100 at the sbm sites to the membrane pore, through which the single-stranded DNA is transferred to the recipient. sbmC and sbmD, each of which contained a characteristic inverted repeat sequence, overlapped with the promoter region for the traM gene. This suggests that the expression of the traM gene may be regulated by its own product.     

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.     

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.     

IHF protein inhibits cleavage but not assembly of plasmid R388 relaxosomes

Relaxosomes are specific nucleoprotein structures involved in DNA-processing reactions during bacterial conjugation. In this work, we present evidence indicating that plasmid R388 relaxosomes are composed of origin of transfer (oriT) DNA plus three proteins TrwC relaxase, TrwA nic-cleavage accessory protein and integration host factor (IHF), which acts as a regulatory protein. Protein IHF bound to two sites (ihfA and ihfB) in R388 oriT, as shown by gel retardation and DNase I footprinting analysis. IHF binding in vitro was found to inhibit nic-cleavage, but not TrwC binding to supercoiled DNA. However, no differences in the frequency of R388 conjugation were found between IHF- and IHF+ donor strains. In contrast, examination of plasmid DNA obtained from IHF- strains revealed that R388 was obtained mostly in relaxed form from these strains, whereas it was mostly supercoiled in IHF+ strains. Thus, IHF could have an inhibitory role in the nic-cleavage reaction in vivo. It can be speculated that triggering of conjugative DNA processing during R388 conjugation can be mediated by IHF release from oriT.     

Site-specific recombination at oriT of plasmid R1162 in the absence of conjugative transfer.

R1162 is efficiently comobilized during conjugative transfer of the self-transmissible plasmid R751. Bacteriophage M13 derivatives that contain two directly repeated copies of oriT, the site on R1162 DNA required in cis for mobilization, were constructed. Phage DNA molecules underwent recombination during infection of Escherichia coli, with the product retaining a single functional copy of oriT. Recombination was strand specific and depended on R1162 gene products involved in mobilization, but did not require the self-transmissible plasmid vector. Two genes were identified, one essential for recombination and the other affecting the frequency of recombination. Recombination of bacteriophage DNA could form the basis of a simple model for some of the events occurring during conjugation without the complexity of a true mating system.     

Mutational and physical analysis of F plasmid traY protein binding to oriT

F plasmid traY protein binding to wild-type or deleted regions containing the TraY-binding site, sbyA, was studied in vitro. The principal DNA-protein complex was formed with DNA segments including the sbyA site defined by footprinting and (with lesser affinity) with truncated segments that retained the leftward two-thirds of sbyA. This located the major sequence determinants for TraY binding between bp 204 and 227 on the oriT map. For all sequences tested, bound TraY induced bending of approximateiy 50 to 55°, and centred between bp 214 and 221. Thermodynamic and mobility analyses indicated that two TraY protomers bind to sbyA. At higher TraY concentrations, additional TraY bound to the left of the sbyA in a region previously shown to bind IHF (site IHF A). TraY binding to this additional site (sbyC) was inhibited by IHF. Sequence similarities shared by sbyA, sbyB, and SbyC may include the critical base pairs for TraY binding.     

The TraM protein of plasmid R1 is a DNA-binding protein

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies. Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist. The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.     

NikAB- or NikB-dependent intracellular recombination between tandemly repeated oriT sequences of plasmid R64 in plasmid or single-stranded phage vectors

The origin of transfer (oriT) of a bacterial plasmid plays a key role in both the initiation and termination of conjugative DNA transfer. We have previously shown that a conjugation-dependent recombination between the tandem R64 oriT sequences cloned into pHSG398 occurred, resulting in the deletion of the intervening sequence during DNA transfer. In this study, we tandemly cloned two oriT sequences of IncI1 plasmid R64 into pUC18. Specific recombination between the two oriT sequences in pUC18 was observed within Escherichia coli cells harboring mini-R64. This recombination was found to be independent of both the recA gene and conjugative DNA transfer. The R64 genes nikA and nikB, required for conjugal DNA processing, were essential for this recombination. Although a fully active 92-bp oriT sequence was required at one site for the recombination, the 44-bp oriT core sequence was sufficient at the other site. Furthermore, when two oriT sequences were tandemly cloned into the single-stranded phage vector M13 and propagated within E. coli cells, recombination between the two oriT sequences was observed, depending on the nikB gene. These results suggest that the R64 relaxase protein NikB can execute cleavage and rejoining of single-stranded oriT DNA within E. coli cells, whereas such a reaction in double-stranded oriT DNA requires collaboration of the two relaxosome proteins, NikA and NikB.     

Mobilization of chimeric oriT plasmids by F and R100-1: role of relaxosome formation in defining plasmid specificity

Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).     

Site- and strand-specific nicking in vitro at oriT by the traY-traI endonuclease of plasmid R100.

We developed an in vitro system to reproduce a site- and strand-specific nicking at the oriT region of plasmid R100. The nicking reaction was dependent on the purified TraY protein and on the lysate, which was prepared from cells overproducing the TraI protein. This supports the idea that the protein products of two genes, traY and traI, constitute an endonuclease that introduces a specific nick in vivo in the oriT region of the conjugative plasmids related to R100. The products were the "complex" DNA molecules with a protein covalently linked with the 5'-end of the nick. The nick was introduced in the strand, which is supposed to be transferred to recipient cells during conjugation, and was located at the site 59 base pairs upstream of the TraY protein binding site, sbyA.     

Transfer region of IncI1 plasmid R64 and role of shufflon in R64 transfer.

To locate the transfer region of the 122-kiloase plasmid R64drd-11 belonging to incompatibility group I1, a series of deletion derivatives was constructed by in vitro recombinant DNA techniques followed by double homologous recombination in vivo. A plasmid designated pKK609 and bearing a 56.7-kilobase R64 sequence was the smallest transferable plasmid. A plasmid designated pKK610 and no longer possessing the 44-base-pair sequence of the R64 transfer system is located at one end. The other end of the R64 transfer region comprises a DNA segment of about 19 kilobases responsible for pilus formation. Shufflon, DNA with a novel rearrangement in R64, was found to be involved in pilus formation.     

Deletion analysis of the F plasmid oriT locus.

Functional domains of the Escherichia coli F plasmid oriT locus were identified by deletion analysis. DNA sequences required for nicking or transfer were revealed by cloning deleted segments of oriT into otherwise nonmobilizable pUC8 vectors and testing for their ability to promote transfer or to be nicked when tra operon functions were provided in trans. Removal of DNA sequences to the right of the central A + T-rich region (i.e., from the direction of traM) did not affect the susceptibility of oriT to nicking functions; however, transfer efficiency for oriT segments deleted from the right was progressively reduced over an 80- to 100-bp interval. Deletions extending toward the oriT nick site from the left did not affect the frequency of transfer if deletion endpoints lay at least 22 bp away from the nick site. Deletions or insertions in the central, A + T-rich region caused periodic variation in transfer efficiency, indicating that phase relationships between nicking and transfer domains of oriT must be preserved for full oriT function. These data show that the F oriT locus is extensive, with domains that individually contribute to transfer, nicking, and overall structure.     

Initiation and termination of DNA transfer at F plasmid oriT

DNA sequences within the F plasmid transfer origin (oriT) were tested for their ability to initiate or terminate conjugal transfer. Mutant and wild-type oriT elements were cloned as direct repetitions flanking the rpsL gene on a pBR322-based plasmid, and the frequency of deletion of this segment during matings sponsored by F’lac (F42) with streptomycin-resistant recipients was measured. Shortened oriT elements that lacked adjacent TraM-binding sites allowed efficient initiation and termination. Some truncated orir segments lacking the TraM-binding sites and the TraY-binding site, sbyA, initiated transfer inefficiently, but nevertheless promoted efficient termination. Removal of TraM-, TraY-, and IHF-binding sites severely reduced both nicking and termination. Point mutations that previously had been reported to prevent nicking caused reduced levels of both initiation and termination. These results indicate that regions of oriT supporting initiation are more extensive than those needed for termination, although some regions are required for both. Moreover, termination can be effective for some mutant loci that do not support efficient nicking.     

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC.

Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA. This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation. This region contains three genes, trwA, trwB, and trwC. By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination. Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination. TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process. Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases. This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required. This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.     

Specific binding of the NikA protein to one arm of 17-base-pair inverted repeat sequences within the oriT region of plasmid R64.

Products of the nikA and nikB genes of plasmid R64 have been shown to form a relaxation complex with R64 oriT DNA and to function together as an oriT-specific nickase. We purified the protein product of the nikA gene. The purified NikA protein bound specifically to the oriT region of R64 DNA. Gel retardation assays and DNase I footprinting analyses indicated that the NikA protein bound only to the right arm of 17-bp inverted repeat sequences; the right arm differed from the left arm by a single nucleotide. The binding site is proximal to the nick site and within the 44-bp oriT core sequence. Binding of the NikA protein induced DNA bending within the R64 oriT sequence.