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1.
Evidence that DNA helicase I and oriT site-specific nicking are both functions of the F TraI protein 总被引:14,自引:0,他引:14
Site-specific and strand-specific nicking at the origin of transfer (oriT) of the F sex factor is the initial step in conjugal DNA metabolism. Then, DNA helicase I, the product of the traI gene, processively unwinds the plasmid from the nick site to generate the single strand of DNA that is transferred to the recipient. The nick at oriT is produced by the combined action of two Tra proteins, TraY and TraZ. The traZ gene was never precisely mapped, as no available point mutation uniquely affected TraZ-dependent oriT nicking. With several new mutations, we have demonstrated that TraZ activity is dependent upon traI DNA sequences. The simplest interpretation of this finding is that the F TraI protein is bifunctional, with DNA unwinding and site-specific DNA nicking activities. 相似文献
2.
Characterization of the reaction product of the oriT nicking reaction catalyzed by Escherichia coli DNA helicase I. 总被引:8,自引:2,他引:6
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DNA helicase I, encoded on the Escherichia coli F plasmid, catalyzes a site- and strand-specific nicking reaction within the F plasmid origin of transfer (oriT) to initiate conjugative DNA strand transfer. The product of the nicking reaction contains a single phosphodiester bond interruption as determined by single-nucleotide resolution mapping of both sides of the nick site. This analysis has demonstrated that the nick is located at precisely the same site previously shown to be nicked in vivo (T. L. Thompson, M. B. Centola, and R. C. Deonier, J. Mol. Biol. 207:505-512, 1989). In addition, studies with two oriT point mutants have confirmed the specificity of the in vitro reaction. Characterization of the nicked DNA product has revealed a modified 5' end and a 3' OH available for extension by E. coli DNA polymerase I. Precipitation of nicked DNA with cold KCl in the presence of sodium dodecyl sulfate suggests the existence of protein covalently attached to the nicked DNA molecule. The covalent nature of this interaction has been directly demonstrated by transfer of radiolabeled phosphate from DNA to protein. On the basis of these results, we propose that helicase I becomes covalently bound to the 5' end of the nicked DNA strand as part of the reaction mechanism for phosphodiester bond cleavage. A model is presented to suggest how helicase I could nick the F plasmid at oriT and subsequently unwind the duplex DNA to provide single-stranded DNA for strand transfer during bacterial conjugation. 相似文献
3.
In vitro cleavage of double- and single-stranded DNA by plasmid RSF1010-encoded mobilization proteins. 总被引:29,自引:2,他引:27
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We have used purified RSF1010 mobilization proteins to reproduce in vitro a strand-specific nicking at the plasmid origin of transfer, oriT. In the presence of Mg2+, the proteins MobA (78-kDa form of RSF1010 DNA primase), MobB, and MobC and supercoiled or linear duplex oriT DNA form large amounts of a cleavage complex, which is characterized by its sensitivity to protein-denaturant treatment. Upon addition of SDS to such a complex, a single strand break is generated in the DNA, and MobA is found linked to the 5' nick terminus, presumably covalently. The double-strand nicking activity of MobA requires, in addition to Mg2+, the presence of MobC and is stimulated by the presence of MobB. The nick site has been shown by DNA sequencing to lie at the position cleaved in vivo during transfer, between nucleotides 3138/3139 in the r strand of RSF1010. We have found that MobA will also cleave DNA at sites other than oriT if the DNA is present in single-stranded form. Breakage in this case occurs in the absence of denaturing conditions, and after prolonged incubation, reclosure can be demonstrated. 相似文献
4.
The product of the Escherichia coli F plasmid traI gene is required for DNA transfer via bacterial conjugation. This bifunctional protein catalyzes the unwinding of duplex DNA and is a sequence-specific DNA transesterase. The latter activity provides the site- and strand-specific nick required to initiate DNA transfer. To address the role of the TraI helicase activity in conjugative DNA transfer traI mutants were constructed and their function in DNA transfer was evaluated using genetic and biochemical methods. A traI deletion/insertion mutant was transfer-defective as expected. A traI C-terminal deletion that removed the helicase-associated motifs was also transfer-defective despite the fact that the region of traI encoding the transesterase activity was intact. Biochemical studies demonstrated that the N-terminal domain was sufficient to catalyze oriT-dependent transesterase activity. Thus, a functional transesterase was not sufficient to support DNA transfer. Finally, a point mutant, TraI-K998M, that lacked detectable helicase activity was characterized. This protein catalyzed oriT-dependent transesterase activity in vitro and in vivo but failed to complement a traI deletion strain in conjugative DNA transfer assays. Thus, both the transesterase and helicase activities of TraI are essential for DNA strand transfer. 相似文献
5.
Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids. 总被引:5,自引:2,他引:3
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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. 相似文献
6.
Escherichia coli DNA helicase I. Characterization of the protein and of its DNA-binding properties 总被引:4,自引:0,他引:4
Gene traI of the Escherichia coli F sex factor which encodes DNA helicase I was subcloned in a lambda pL-based plasmid vector and expressed in a background of pL non-repressing cells. Neither the non-repressed pL promoter nor the production of a high level of functional helicase I are toxic. Enzyme purified from this source was studied in the electron microscope. The results show that helicase I binds cooperatively to single-stranded DNA. DNA covered with the helicase appears in fixed, negatively stained specimens as a smooth-contoured filament with a diameter of 12.5 +/- 0.4 nm and an axial periodicity of 7.0 +/- 0.2 nm. In unfixed specimens, discrete particles with axes of 12.7 +/- 0.5 nm and 7.2 +/- 0.5 nm are visible. They are consistent in size with helicase I monomers (Mr 180,000) suggesting that the molecule is almost isometric, despite a frictional ratio of 1.71 calculated from its diffusion coefficient. Helicase I free of DNA appears as aggregates. For comparison, a truncated traI, lacking coding for the amino-terminus of the product, was cloned by fusing it to an MS2 replicase gene fragment. The chimeric gene product (named helicase I del29) retains strand-separating activity although it fails to show cooperative DNA binding behavior. Judged from the length of the helicase-I-specific sequence of this polypeptide, traI is located 1.3 kb nearer to the distal end of the F transfer operon compared to the position proposed in a previous genetic map. The revised location of traI has implications for understanding distal functions of the transfer operon. 相似文献
7.
Location of the nick at oriT of the F plasmid 总被引:16,自引:0,他引:16
The oriT locus of the Escherichia coli K12 F plasmid contains a site at which one of the DNA strands is cleaved as a prelude to conjugal transmission to recipient bacteria. We have remapped this site biochemically by using oriT-containing plasmids that were purified from bacteria expressing the F transfer (tra) functions. The strand interruption was found on the transferred strand 137 base-pairs clockwise of the center of the BglII site at 66.7 on the F map. This location is consistent with the locations anticipated from studies of delta traF' plasmids, but it differs from previous results by other investigators. The strand interruption produced a 3'-OH, but the nature of the 5' terminus of the strand on the other side of the nick was not determined. Some DNA sequence motifs in the vicinity of the oriT nick site of F resemble the chromosomal site involved in formation of delta traF'purE plasmids. 相似文献
8.
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. 相似文献
9.
Byrd DR Sampson JK Ragonese HM Matson SW 《The Journal of biological chemistry》2002,277(45):42645-42653
TraI (DNA helicase I) is an Escherichia coli F plasmid-encoded 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 approximately 650 residues between the transesterase domain and the helicase-associated motifs. This region is required for helicase activity. 相似文献
10.
Escherichia coli DNA helicase I catalyzes a site- and strand-specific nicking reaction at the F plasmid oriT. 总被引:5,自引:0,他引:5
A site- and strand-specific nick, introduced in the F plasmid origin of transfer, initiates conjugal DNA transfer during bacterial conjugation. Recently, molecular genetic studies have suggested that DNA helicase I, which is known to be encoded on the F plasmid, may be involved in this nicking reaction (Traxler, B. A., and Minkley, E. G., Jr. (1988) J. Mol. Biol. 204, 205-209). We have demonstrated this site- and strand-specific nicking event using purified helicase I in an in vitro reaction. The nicking reaction requires a superhelical DNA substrate containing the F plasmid origin of transfer, Mg2+ and helicase I. The reaction is protein concentration-dependent but, under the conditions used, only 50-70% of the input DNA substrate is converted to the nicked species. Genetic data (Everett, R., and Willetts, N. (1980) J. Mol. Biol. 136, 129-150) have also suggested the involvement of a second F-encoded protein, the TraY protein, in the oriT nicking reaction. Unexpectedly, the in vitro nicking reaction does not require the product of the F plasmid traY gene. The implications of this result are discussed. The phosphodiester bond interrupted by helicase I has been shown to correspond exactly to the site nicked in vivo suggesting that helicase I is the site- and strand-specific nicking enzyme that initiates conjugal DNA transfer. Thus, helicase I is a bifunctional protein which catalyzes site- and strand-strand specific nicking of the F plasmid in addition to the previously characterized duplex DNA unwinding (helicase) reaction. 相似文献
11.
TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic. 相似文献
12.
H D Bradshaw Jr B A Traxler E G Minkley Jr E W Nester M P Gordon 《Journal of bacteriology》1990,172(7):4127-4131
A 6.9-kilobase region of the Escherichia coli F plasmid containing the 3' half of the traD gene and the entire traI gene (encodes the TraI protein, DNA helicase I and TraI, a polypeptide arising from an internal in-frame translational start in traI) has been sequenced. A previously unidentified open reading frame (tentatively trbH) lies between traD and traI. 相似文献
13.
The TraI protein has two essential roles in transfer of conjugative plasmid F Factor. As part of a complex of DNA-binding proteins, TraI introduces a site- and strand-specific nick at the plasmid origin of transfer (oriT), cutting the DNA strand that is transferred to the recipient cell. TraI also acts as a helicase, presumably unwinding the plasmid strands prior to transfer. As an essential feature of its nicking activity, TraI is capable of binding and cleaving single-stranded DNA oligonucleotides containing an oriT sequence. The specificity of TraI DNA recognition was examined by measuring the binding of oriT oligonucleotide variants to TraI36, a 36-kD amino-terminal domain of TraI that retains the sequence-specific nucleolytic activity. TraI36 recognition is highly sequence-specific for an 11-base region of oriT, with single base changes reducing affinity by as much as 8000-fold. The binding data correlate with plasmid mobilization efficiencies: plasmids containing sequences bound with lower affinities by TraI36 are transferred between cells at reduced frequencies. In addition to the requirement for high affinity binding to oriT, efficient in vitro nicking and in vivo plasmid mobilization requires a pyrimidine immediately 5' of the nick site. The high sequence specificity of TraI single-stranded DNA recognition suggests that despite its recognition of single-stranded DNA, TraI is capable of playing a major regulatory role in initiation and/or termination of plasmid transfer. 相似文献
14.
Characterization of the Escherichia coli F factor traY gene product and its binding sites. 总被引:2,自引:1,他引:1
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The traY gene product (TraYp) from the Escherichia coli F factor has previously been purified and shown to bind a DNA fragment containing the F plasmid oriT region (E. E. Lahue and S. W. Matson, J. Bacteriol. 172:1385-1391, 1990). To determine the precise nucleotide sequence bound by TraYp, DNase I footprinting was performed. The TraYp-binding site is near, but not coincident with, the site that is nicked to initiate conjugative DNA transfer. In addition, a second TraYp binding site, which is coincident with the mRNA start site at the traYI promoter, is described. The Kd for each binding site was determined by a gel mobility shift assay. TraYp exhibits a fivefold higher affinity for the oriT binding site compared with the traYI promoter binding site. Hydrodynamic studies were performed to show that TraYp is a monomer in solution under the conditions used in DNA binding assays. Early genetic experiments implicated the traY gene product in the site- and strand-specific endonuclease activity that nicks at oriT (R. Everett and N. Willetts, J. Mol. Biol. 136:129-150, 1980; S. McIntire and N. Willetts, Mol. Gen. Genet. 178:165-172, 1980). As this activity has recently been ascribed to helicase I, it was of interest to see whether TraYp had any effect on this reaction. Addition of TraYp to nicking reactions catalyzed by helicase I showed no effect on the rate or efficiency of oriT nicking. Roles for TraYp in conjugative DNA transfer and a possible mode of binding to DNA are discussed. 相似文献
15.
Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport. 总被引:15,自引:9,他引:6
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The DNA transfer stage of conjugation requires the products of the F sex factor genes traMYDIZ and the cis-acting site oriT. Previous interpretation of genetic and protein analyses suggested that traD, traI, and traZ mapped as contiguous genes at the distal end of the transfer operon and saturated this portion of the F transfer region (which ends with an IS3 element). Using antibodies prepared against the purified TraD and TraI proteins, we analyzed the products encoded by a collection of chimeric plasmids constructed with various segments of traDIZ DNA. We found the traI gene to be located 1 kilobase to the right of the position suggested on previous maps. This creates an unsaturated space between traD and traI where unidentified tra genes may be located and leaves insufficient space between traI and IS3 for coding the 94-kilodalton protein previously thought to be the product of traZ. We found that the 94-kilodalton protein arose from a translational restart and corresponds to the carboxy terminus of traI; we named it TraI*. The precise physical location of the traZ gene and the identity of its product are unknown. The oriT nicking activity known as TraZ may stem from unassigned regions between traD and traI and between traI and IS3, but a more interesting possibility is that it is actually a function of traI. On our revised map, the position of a previously detected RNA polymerase-binding site corresponds to a site at the amino terminus of traI rather than a location 1 kilobase into the coding region of the gene. Furthermore, the physical and genetic comparison of the F traD and traI genes with those of the closely related F-like conjugative plasmids R1 and R100 is greatly simplified. The translational organization we found for traI, together with its identity as the structural gene for DNA helicase I, suggests a possible functional link to several other genes from which translational restart polypeptides are expressed. These include the primases of the conjugative plasmids ColI and R16, the primase-helicase of bacteriophage T7, and the cisA product (nickase) of phage phi X174. 相似文献
16.
The F plasmid origin of transfer: DNA sequence of wild-type and mutant origins and location of origin-specific nicks. 总被引:24,自引:1,他引:23
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The DNA sequence of the F plasmid origin of conjugal DNA transfer, oriT , has been determined. The origin lies in an intercistronic region which contains several inverted repeat sequences and a long AT-rich tract. Introduction of a nick into one of the DNA strands in the oriT region precedes the initiation of conjugal DNA replication, and the position of the strand-specific nicks acquired by a lambda oriT genome upon propagation in Flac-carrying cells has been determined. The nicks were not uniquely positioned, rather there was a cluster of three major and up to 20 minor sites: the biological significance of this observation is not yet fully clear. Nine independent point mutations which inactivate oriT function have been sequenced and found to alter one or other of two nucleotide positions which lie 14 and 19 bp to one side of the rightmost (as drawn) major nick site. These key nucleotides may lie in a recognition sequence for the oriT endonuclease, since mutations at these sites prevent nicking at oriT . 相似文献
17.
Electron microscopy was used to characterize the DNA-unwinding reaction catalysed by Escherichia coli DNA helicase I. Linear DNA with 5'-protruding strands as well as single-stranded gaps was incubated, under unwinding assay conditions, with the helicase. E. coli single-stranded-DNA-binding protein (SSB) was added to order the denatured DNA. Up to 70% of the sites of SSB-complexed DNA were observed as forks. The position of the strand-separating enzyme was indicated by a gap in the complex between fork and SSB on that arm which initially provided the binding site. The complex between DNA and helicase varied in length although in all cases it was long enough to comprise several helicase I molecules. A mutant helicase I (helicase I del29) which, unlike the wild-type enzyme, fails to show cooperative DNA-binding behaviour was found to prevent an abnormally short stretch of DNA near the fork from binding SSB. Apparently, one or very few helicase molecules would be sufficient for the opening of a DNA duplex although, typically, the fork is shifted by a tract of helicase I molecules. SSB displaces helicase I from single-stranded DNA but fails to do so from a fork or a single-strand/double-strand junction. The difference is consistent with the observation that SSB does not inhibit the unwinding reaction despite its rapid association with the separated strands. Helicase I unwinds in the 5'-3' direction of the bound strand. Observations so far indicate that the enzyme exploits the single strand at the initial DNA-binding site for orienting its action, and not the complementary, completely base-paired strand. 相似文献
18.
pED806 , a pBR322 derivative carrying the origin of transfer ( oriT ) of F, was rapidly lost from cells carrying an F tra+ plasmid. Instability was increased in a RecA- host, and depended in particular upon the Ftra YZ genes that produce the nick at oriT at which transfer is initiated. Instability was also correlated with the orientation of the oriT fragment in the vector plasmid. Mutants of pED806 selected as being stable in the presence of Flac proved to carry cis-dominant oriT mutations. The oriT site was subcloned from pED806 on a HaeII fragment including a HaeII-Bg/II segment of F DNA approximately 385 base pair (bp) long into the 2.25 kilobase (kb) vector plasmid pED825 , giving pED822 . pED822 was fully proficient for oriT function, and recircularised in recipient cells by a recA- and tra-independent oriT -specific ligation/recombination event. ' Phasmids ' constructed by cloning pED806 or an oriT - mutant into a lambda vector were used to confirm that the nick site in lambda oriT phages grown in the presence of Flac tra+ is indeed at oriT . The nick site in a further lambda oriT phage (ED lambda 102) was then located 140 +/- 20 bp from the Bg/II site forming one terminus of the F fragment cloned in pED806 and pED822 . 相似文献
19.
Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli 总被引:13,自引:0,他引:13
A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor. Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity. This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa. The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor. The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay. The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction. The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity. In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently. The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein. However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria. The discovery of a new helicase brings the total number of helicases found in E. coli cell extracts (lacking F factor) to five. 相似文献
20.
DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair. 相似文献