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1.
The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA, RNA hybrid duplexes and DNA-DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.  相似文献   

2.
Helicase I has been purified to greater than 95% homogeneity from an F+ strain of Escherichia coli, and characterized as a single-stranded DNA-dependent ATPase and a helicase. The duplex DNA unwinding reaction requires a region of ssDNA for enzyme binding and concomitant nucleoside 5'-triphosphate hydrolysis. All eight predominant nucleoside 5'-triphosphates can satisfy this requirement. Unwinding is unidirectional in the 5' to 3' direction. The length of duplex DNA unwound is independent of protein concentration suggesting that the unwinding reaction is highly processive. Kinetic analysis of the unwinding reaction indicates that the enzyme turns over very slowly from one DNA substrate molecule to another. The ATP hydrolysis reaction is continuous when circular partial duplex DNA substrates are used as DNA effectors. When linear partial duplex substrates are used ATP hydrolysis is barely detectable, although the kinetics of the unwinding reaction on linear partial duplex substrates are identical to those observed using a circular partial duplex DNA substrate. This suggests that ATP hydrolysis fuels continuous translocation of helicase I on circular single-stranded DNA while on linear single stranded DNA the enzyme translocates to the end of the DNA molecule where it must slowly dissociate from the substrate molecule and/or slowly associate with a new substrate molecule, thus resulting in a very low rate of ATP hydrolysis.  相似文献   

3.
A single-stranded DNA-dependent ATPase from monkey kidney tissue culture cells (CV-1) has been found associated with SV40 chromatin. This ATPase activity is distinguishable from the ATPase activity of T-antigen by the following properties: the Km for ATP, elution from phosphocellulose, and stimulation of the ATPase activity by single-stranded DNA but not by double-stranded DNA. The ATPase has been isolated and characterized from the nuclei of uninfected cells. ATP hydrolysis is dependent on single-stranded DNA and a divalent cation. The km values for ATP and single-stranded DNA are 0.024 mM and 0.09 microgram/ml, respectively. The affinity of the ATPase for single-stranded DNA is sufficiently high that the enzyme co-sediments with single-stranded DNA in glycerol gradients. The binding of single-stranded DNA is independent of ATP and MgCl2; however, ATP hydrolysis increases the exchange of enzyme between different DNA molecules. Form I (superhelical) SV40 DNA is also a substrate for ATPase binding, but relaxed Form I, Form II (nicked circular), and double-stranded linear SV40 DNAs are not substrates. Because the DNA helix within chromatin is not under the same kind of tortional strain as Form I DNA, we hypothesize that the ATPase is bound to the single-stranded regions of replication forks in the SV40 chromatin.  相似文献   

4.
A single-stranded DNA-dependent ATP gamma-phosphohydrolase of Mr 56000 induced after infection of Escherichia coli cells with bacteriophage T4, probably the ATPase dependent on gene dda of the phage, was isolated. Studies on the enzyme show that in the presence of ATP and M2+ ions it is capable of dissociating partially double-stranded fd bacteriophage DNA into the single strands and that some 3000 enzyme copies are required to unwind the 6400-nucleotides-long DNA. Unwinding is inhibited by reducing the length of the single-stranded portion of DNA to two nucleotides. In addition it can be inhibited by sulfhydryl reagents which block the ATPase or by trapping free enzyme molecules in the assay system. The results suggest that unwinding is initiated near the single-stranded portion of the DNA and is driven by the ATPase. It further appears that the enzyme unwinds by adsorbing to the DNA. Affinity of the enzyme for double-standed DNA is not detectable by DNA binding assay.  相似文献   

5.
The ATP-dependent DNase from Hemophilus influenzae digests double-stranded linear DNA molecules exonucleolytically while hydrolyzing large amounts of ATP to ADP. Various cross-linked linear duplex DNA molecules are partially resistant to the exonuclease action. Vaccinia DNA, containing natural terminal cross-links (probably in the form of terminal single-stranded loops), is much more slowly degraded than comparable "open-ended" DNA molecules, and ATP is consumed at a proportionately lower rate. It is postulated that the vaccinia DNA molecules undergo slow terminal cleavage by the single strand specific endonuclease activity of the enzyme, and are then rapidly degraded by the double strand exonuclease activity. Phage T7 DNA, containing an average of 100 4',5'8-trimethylpsoralen cross-links/molecule at random internal sites, is digested only to the extent of 2 to 3%. However, ATP hydrolysis continues at a linear rate long after DNA digestion has ceased. A stable enzyme-DNA complex is formed as demonstrated by co-sedimentation of DNA and ATPase activity in sucrose gradients. The hypothesis is advanced that the enzyme digests exonucleolytically to the first cross-link at each end of the DNA molecules where further movement is prevented. The enzyme then remains bound at the cross-links and functions continuously as an ATPase.  相似文献   

6.
Purification of a RecA protein analogue from Bacillus subtilis   总被引:29,自引:0,他引:29  
We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.  相似文献   

7.
T4 phage gene uvsX product catalyzes homologous DNA pairing.   总被引:12,自引:2,他引:10       下载免费PDF全文
Gene uvsX of phage T4 controls genetic recombination and the repair of DNA damage. We have recently purified the gene product, and here describe its properties. The protein has a single-stranded DNA-dependent ATPase activity. It binds efficiently to single- and double-stranded DNAs at 0 degrees C in a cooperative manner. At 30 degree C the double-stranded DNA-protein complex was stable, but the single-stranded DNA-protein complex dissociated rapidly. The instability of the latter complex was reduced by ATP. The protein renatured heat-denatured double-stranded DNA, and assimilated linear single-stranded DNA into homologous superhelical duplexes to produce D-loops. The reaction is stimulated by gene 32 protein when the uvsX protein is limiting. With linear double-stranded DNA and homologous, circular single-stranded DNA, the protein catalyzed single-strand displacement in the 5' to 3' direction with the cooperation of gene 32 protein. All reactions required Mg2+, and all except DNA binding required ATP. We conclude that the uvsX protein is directly involved in strand exchange and is analogous to the recA protein of Escherichia coli. The differences between the uvsX protein and the recA protein, and the role of gene 32 protein in single-strand assimilation and single-strand displacement are briefly discussed.  相似文献   

8.
A form of DNA polymerase alpha was purified several thousandfold from a protein extract of Xenopus laevis eggs. The enzyme effectively converts, in the presence of ribonucleoside triphosphates, a circular single-stranded phage fd DNA template into a double-stranded DNA form and, therefore, must be associated with a DNA primase. We first show by gel electrophoresis in the presence of sodium dodecyl sulfate that both enzymatic activities, DNA polymerase and primase, most probably reside on a greater than 100 000-Da subunit of the DNA polymerase holoenzyme. We then assayed the polymerase-primase at various template/enzyme ratios and found that the DNA complementary strand sections synthesized in vitro belong to defined size classes in the range of 600-2000 nucleotides, suggesting preferred start and/or stop sites on the fd DNA template strand. We show that the stop sites coincide with stable hairpin structures in fd DNA. We have used a fd DNA template, primed by a restriction fragment of known size, to show that the polymerase-primase stops at the first stable hairpin structure upstream from the 3'-OH primer site when the reaction was carried out at 0.1 mM ATP. However, at 2 mM ATP the enzyme was able to travers this and other stop sites on the fd DNA template strand leading to the synthesis of 2-4 times longer DNA strands. Our results suggest a role for ATP in the polymerase-primase-catalyzed chain-elongation reaction.  相似文献   

9.
T Fujiyoshi  J Nakayama  M Anai 《Biochemistry》1982,21(17):4159-4164
The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.  相似文献   

10.
Bacillus subtilis Marburg TI (thy,trpC2) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease MII), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000:1 by comparing the initial rates of introducing single-strand breaks into the DNA's.  相似文献   

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