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141.
Escherichia coli helicase II (uvrD) protein can completely unwind fully duplex linear and nicked circular DNA 总被引:12,自引:0,他引:12
We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
142.
We have investigated the association kinetics of the co-operatively binding T4-coded gene 32 (helix destabilizing) protein with a variety of single-stranded homopolynucleotides (both RNA and DNA). Stopped-flow mixing experiments were performed by monitoring the partial quenching of the intrinsic tryptophan fluorescence of the protein upon binding to the nucleic acid under conditions where the nucleic acid concentration is in great excess over the protein concentration. Investigations of the association rate (and rate constants) as a function of solution variables has demonstrated quite different behavior at the extremes of “low” and “high” salt concentration. Under low salt (high binding constant) conditions the non-co-operative association is rate-limiting and we measure a bimolecular rate constant of 3 × 106 to 4 × 106 m?1 (nucleotide)s?1 (0·1 m-NaCl, 25·0 °C). However, at higher salt concentrations (lower binding constant) a pre-equilibrium involving non-co-operatively bound protein is established, followed by the rate-limiting formation of co-operatively bound protein clusters.Based on these observations we have proposed a mechanism for the formation of co-operatively bound T4 gene 32 protein clusters, under conditions of low binding density, which consists of three steps: (1) pre-equilibrium formation of non-co-operatively bound protein (nucleation); followed by (2) association of free protein to the singly contiguous sites established in the nucleation step, hence forming the first co-operative interactions (growth step); and (3) a redistribution of the growing protein clusters to form the final equilibrium distribution. From comparisons of our experimental values of the forward rate constant for the second step (growth of clusters) with theoretical estimates based on the work of Berg &; Blomberg (1976,1978) we infer that the T4 gene 32 protein is able to translocate along singlestranded polynucleotides. The implications of these results for the in vivo action of the T4 gene 32 protein are discussed. 相似文献
143.
144.
A directional, high-frequency chromosomal mobilization system for genetic mapping of Rhizobium meliloti.
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A system for mapping of the Rhizobium meliloti chromosome that utilizes transposon Tn5-Mob, which carries the mobilization site of IncP plasmid RP4 (R. Simon, Mol. Gen. Genet. 196:413-420, 1984), was developed. Insertions of Tn5-Mob that were located at particular sites on the R. meliloti chromosome were isolated and served as origins of high-frequency chromosomal transfer when IncP tra functions were provided in trans. This approach is, in principle, applicable to any gram-negative bacterium in which Tn5 can transpose and into which IncP plasmids can conjugate. 相似文献
145.
Immunochemical quantitation of adducts induced in DNA by cis-diamminedichloroplatinum (II) and analysis of adduct-related DNA-unwinding 总被引:2,自引:0,他引:2
A M Fichtinger-Schepman R A Baan A Luiten-Schuite M van Dijk P H Lohman 《Chemico-biological interactions》1985,55(3):275-288
Antibodies elicited against the haptens cis-Pt(NH3)2dGuodGMP and its ribo-analog, both covalently coupled to bovine serum albumin, recognize adducts of cis-diamminedichloroplatinum(II) (cis-DDP) in DNA. Antibody-binding to cis-DDP-DNA strongly depends on the accessibility of the adducts to the antibodies. In double-stranded cis-DDP-DNA with low Pt: nucleotide ratios (rb's), this accessibility is enhanced by unwinding of the cis-DDP-DNA, e.g. by heat-denaturation. An unwinding effect is also induced by the cis-DDP treatment itself. A260nm readings of cis-DDP-DNA samples indicate an increased denaturation of the DNA at increasing Pt-contents. The data obtained after heat-denaturation of the same samples show a growing capability to renaturation when the rb-values increase from 0 to 0.04; at 0.04 less than rb less than 0.18 the renaturation effect gradually disappears. In the competitive enzyme-linked immunosorbent assay (ELISA), the cis-DDP-adducts in heat-denatured DNA are detected in the pmol range; in DNA-digests, however, they are recognized in fmol amounts. For the individual Pt-containing (oligo)nucleotides the amounts causing 50% inhibition in the ELISA were established for the two anti-sera; they were 13.3 +/- 3.8 (fmol +/- S.D.) and 5.4 +/- 1.8 for cis-Pt(NH3)2d(GMP)2; 15.5 +/- 5.4 and 4.0 +/- 1.5 for cis-Pt(NH3)2d(pGpG); (2.6 +/- 1.1) X 10(3) and (2.0 +/- 1.0) X 10(3) for cis-Pt(NH3)2d(pApG); (5.6 +/- 1.9) X 10(3) and (2.9 +/- 0.4) X 10(3) for Pt(NH3)3dGMP. Pt-adducts in a trans-DDP-DNA digest are recognized in pmol amounts and dGMP in nmol quantitatives. Finally, the usefulness of these antibodies for the detection and quantitation of individual cis-DDP-adducts in cis-DDP-DNA digests was demonstrated. 相似文献
146.
Two binding modes in Escherichia coli single strand binding protein-single stranded DNA complexes. Modulation by NaCl concentration 总被引:17,自引:0,他引:17
The binding properties of the Escherichia coli encoded single strand binding protein (SSB) to a variety of synthetic homopolynucleotides, as well as to single stranded M13 DNA, have been examined as a function of the NaCl concentration (25.0 degrees C, pH 8.1). Quenching of the intrinsic tryptophan fluorescence of the SSB protein by the nucleic acid is used to monitor binding. We find that the site size (n) for binding of SSB to all single stranded nucleic acids is quite dependent on the NaCl concentration. For SSB-poly(dT), n = 33 +/- 3 nucleotides/tetramer below 10 mM NaCl and 65 +/- 5 nucleotides/tetramer above 0.20 M NaCl (up to 5 M). Between 10 mM and 0.2 M NaCl, the apparent site size increases continuously with [NaCl]. The extent of quenching of the bound SSB fluorescence by poly(dT) also displays two-state behavior, 51 +/- 3% quenching below 10 mM NaCl and 83 +/- 3% quenching at high [NaCl] (greater than 01.-0.2 M NaCl), which correlates with the observed changes in the occluded site size. On the basis of these observations as well as the data of Krauss et al. (Krauss, G., Sindermann, H., Schomburg, U., and Maass, G. (1981) Biochemistry 20, 5346-5352) and Chrysogelos and Griffith (Chrysogelos, S., and Griffith, J. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,5803-5807) we propose a model in which E. coli SSB binds to single stranded nucleic acids in two binding modes, a low salt mode (n = 33 +/- 3), referred to as (SSB)33, in which the nucleic acid interacts with only two protomers of the tetramer, and one at higher [NaCl], n = 65 +/- 5, (SSB)65, in which the nucleic acid interacts with all 4 protomers of the tetramer. At intermediate NaCl concentrations a mixture of these two binding modes exists which explains the variable site sizes and other apparent discrepancies previously reported for SSB binding. The transition between the two binding modes is reversible, although the kinetics are slow, and it is modulated by NaCl concentrations within the physiological range. We suggest that SSB may utilize both binding modes in its range of functions (replication, recombination, repair) and that in vivo changes in the ionic media may play a role in regulating some of these processes. 相似文献
147.
The interaction of counterions with a suitably long, charged oligomer appears susceptible to treatment in the context of polyelectrolyte theory by the introduction of an end-effect parameter that reflects the reduced association of counterions with the terminal regions of the oligo-ion. Use of a physically reasonable value for the end-effect parameter provides excellent agreement between theory and the experimental data of Elson, Scheffler, and Baldwin [J. Mol. Biol. 54 , 401–415 (1970)] on the dependences of melting temperature on salt concentration and chain length for a series of hairpin helices formed by d(TA) oligomers. The differences in behavior expected for hairpin, dimer, and oligomer-polymer helices are discussed. The salt dependence of the end-joining equilibrium investigated for λ DNA by Wang and Davidson [Cold Spring Harbor Symp. Quant. Biol. 33 , 409–415 (1968)] is treated as an oligomer–polymer interconversion. The dependence of equilibrium constant for this reaction on counterion concentration is in good agreement with that predicted by theory for an end-region totalling 24 nucleotides, the known length of the λ ends. 相似文献
148.
Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA. Using stopped-flow methods we have examined the kinetic mechanism of translocation of UvrD monomers along single-stranded DNA (ssDNA) in vitro by monitoring the transient kinetics of arrival of protein at the 5'-end of the ssDNA. Arrival at the 5'-end was monitored by the effect of protein on the fluorescence intensity of fluorophores (Cy3 or fluorescein) attached to the 5'-end of a series of oligodeoxythymidylates varying in length from 16 to 124 nt. We find that UvrD monomers are capable of ATP-dependent translocation along ssDNA with a biased 3' to 5' directionality. Global non-linear least-squares analysis of the full kinetic time-courses in the presence of a protein trap to prevent rebinding of free protein to the DNA using the methods described in the accompanying paper enabled us to obtain quantitative estimates of the kinetic parameters for translocation. We find that UvrD monomers translocate in discrete steps with an average kinetic step-size, m=3.68(+/-0.03) nt step(-1), a translocation rate constant, kt=51.3(+/-0.6) steps s(-1), (macroscopic translocation rate, mkt=189.0(+/-0.7) nt s(-1)), with a processivity corresponding to an average translocation distance of 2400(+/-600) nt before dissociation (10 mM Tris-HCl (pH 8.3), 20 mM NaCl, 20% (v/v) glycerol, 25 degrees C). However, in spite of its ability to translocate rapidly and efficiently along ssDNA, a UvrD monomer is unable to unwind even an 18 bp duplex in vitro. DNA helicase activity in vitro requires a UvrD dimer that unwinds DNA with a similar kinetic step-size of 4-5 bp step(-1), but an approximately threefold slower unwinding rate of 68(+/-9) bp s(-1) under the same solution conditions, indicating that DNA unwinding activity requires more than the ability to simply translocate directionally along ss-DNA. 相似文献
149.
The SF1 DNA helicases are multi-domain proteins that can unwind duplex DNA in reactions that are coupled to ATP binding and hydrolysis. Crystal structures of two such helicases, Escherichia coli Rep and Bacillus stearothermophilus PcrA, show that the 2B sub-domain of these proteins can be found in dramatically different orientations (closed versus open) with respect to the remainder of the protein, suggesting that the 2B domain is highly flexible. By systematically using fluorescence resonance energy transfer at the single-molecule level, we have determined both the orientation of an E.coli Rep monomer bound to a 3'-single-stranded-double-stranded (ss/ds) DNA junction in solution, as well as the relative orientation of its 2B sub-domain. To accomplish this, we developed a highly efficient procedure for site-specific fluorescence labeling of Rep and a bio-friendly immobilization scheme, which preserves its activities. Both ensemble and single-molecule experiments were carried out, although the single-molecule experiments proved to be essential here in providing quantitative distance information that could not be obtained by steady-state ensemble measurements. Using distance-constrained triangulation procedures we demonstrate that in solution the 2B sub-domain of a Rep monomer is primarily in the "closed" conformation when bound to a 3'-ss/ds DNA, similar to the orientation observed in the complex of PcrA bound to a 3'-ss/ds DNA. Previous biochemical studies have shown that a Rep monomer bound to such a 3'-ss/ds DNA substrate is unable to unwind the DNA and that a Rep oligomer is required for helicase activity. Therefore, the closed form of Rep bound to a partial duplex DNA appears to be an inhibited form of the enzyme. 相似文献
150.
Processive DNA helicases are able to translocate along single-stranded DNA (ssDNA) with biased directionality in a nucleoside triphosphate-dependent reaction, although translocation is not generally sufficient for helicase activity. An understanding of the mechanism of protein translocation along ssDNA requires pre-steady state transient kinetic experiments. Although ensemble experimental approaches have been developed recently for the study of translocation of proteins along DNA, quantitative analysis of the complete time-courses from these experiments, which is needed to obtain quantitative estimates of translocation kinetic parameters (rate constants, processivity, step sizes and ATP coupling) has been lacking. We discuss three ensemble transient kinetic experiments that can be used to study protein translocation along ssDNA, along with the advantages and limitations of each approach. We further describe methods to analyze the complete kinetic time-courses obtained from such experiments performed with a series of ssDNA lengths under "single-round" conditions (i.e. in the absence of re-binding of dissociated protein to DNA). These analysis methods utilize a sequential "n-step" model for protein translocation along ssDNA and enable quantitative determinations of the rate constant, processivity and step size for translocation through global non-linear least-squares fitting of the full time-courses. 相似文献