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1.
RecA protein reinitiates strand exchange on isolated protein-free DNA intermediates. An ADP-resistant process 总被引:8,自引:0,他引:8
Efficient homologous pairing de novo of linear duplex DNA with a circular single strand (plus strand) coated with RecA protein requires saturation and extension of the single strand by the protein. However, strand exchange, the transfer of a strand from duplex DNA to the nucleoprotein filament, which follows homologous pairing, does not require the stable binding of RecA protein to single-stranded DNA. When RecA protein was added back to isolated protein-free DNA intermediates in the presence of sufficient ADP to inhibit strongly the binding of RecA protein to single-stranded DNA, strand exchange nonetheless resumed at the original rate and went to completion. Characterization of the protein-free DNA intermediate suggested that it has a special site or region to which RecA protein binds. Part of the nascent displaced plus strand of the deproteinized intermediate was unavailable as a cofactor for the ATPase activity of RecA protein, and about 30% resisted digestion by P1 endonuclease, which acts preferentially on single-stranded DNA. At the completion of strand exchange, when the distal 5' end of the linear minus strand had been fully incorporated into heteroduplex DNA, a nucleoprotein complex remained that contained all three strands of DNA from which the nascent displaced strand dissociated only over the next 50 to 60 minutes. Deproteinization of this intermediate yielded a complex that also contained three strands of DNA in which the nascent displaced strand was partially resistant to both Escherichia coli exonuclease I and P1 endonuclease. The deproteinized complex showed a broad melting transition between 37 degrees C and temperatures high enough to melt duplex DNA. These results show that strand exchange can be subdivided into two stages: (1) the exchange of base-pairs, which creates a new heteroduplex pair in place of a parental pair; and (2) strand separation, which is the physical displacement of the unpaired strand from the nucleoprotein filament. Between the creation of new heteroduplex DNA and the eventual separation of a third strand, there exists an unusual DNA intermediate that may contain three-stranded regions of natural DNA that are several thousand bases in length. 相似文献
2.
We have isolated a new DNA-dependent ATPase from E. coli. The enzyme has been purified to greater than 90% purity. It appears to be composed of two identical polypeptide chains of molecular weight 20,000. The enzyme catalyzed the hydrolysis of ATP in the presence, but not in the absence, of single-stranded DNA. Double-stranded DNA is not a cofactor. The products of hydrolysis are ADP and Pi. The enzyme also catalyzed strand separation of duplex DNA in the presence of ATP and E. coli DNA binding protein. Two E. coli proteins capable of promoting strand separation have been reported previously and have been termed helicase I and II (Abdel-Monem, M., and Hoffmann-Berling, H. (1977) Eur. J. Biochem. 79, 33-38). Accordingly, this protein is named helicase III. 相似文献
3.
The adenovirus DNA-binding protein (DBP) is a multifunctional protein that is essential for viral DNA replication. DBP binds both single-stranded and double-stranded DNA as well as RNA in a sequence-independent manner. Previous studies showed that DBP does not promote melting of duplex poly(dA-dT) in contrast to prokaryotic single-strand-binding proteins. However, here we show that DBP can displace oligonucleotides annealed to single-stranded M13 DNA. Depending upon the DBP concentration, strands of at least 200 nucleotides can be unwound. Although unwinding of short (17-bp), fully duplex DNA is facilitated by DBP, unwinding of larger (28-bp) duplexes is only possible if single-stranded protruding ends are present. These protruding ends must be at least 4 nucleotides long for optimal unwinding, and both 5' and 3' single-stranded overhangs suffice. DBP-promoted strand displacement is sensitive to MgCl2 and NaCl and not dependent upon ATP. Our results suggest that DBP, through formation of a protein chain on the displaced strand, may destabilize duplex DNA ahead of the replication fork, thereby assisting in strand displacement during replication. 相似文献
4.
The T4 bacteriophage gene 43 (T4 DNA polymerase), 32 (DNA helix-destabilizing protein), and 45 proteins and the complex of the gene 44 and 62 proteins are all required for DNA synthesis beginning at single-stranded breaks in duplex DNA. This synthesis occurs by strand displacement and is not dependent on ribonucleotides, the T4 gene 41 protein, or the T4 initiating protein, each of which is required to begin new chains on single-stranded templates. Electron microscopic analysis shows that duplex molecules with long single-stranded branches are the predominant products of this strand displacement synthesis. 相似文献
5.
Five distinct DNA replicating intermediates have been separated from lysates of bacteriophage G4-infected cells pulse-labelled during the period of replicative form synthesis using propidium diiodide/caesium chloride gradients. These are a partially single-stranded theta structure that is labelled in both the viral and complementary DNA strands; partially single-stranded circles, some with an unfinished viral DNA strand (25%) and some with an unfinished complementary DNA strand (75%); replicative form II(RFII) and replicative form I(RFI) DNA labelled only in the complementary DNA strand. To explain the pulse-label data a model is proposed in which G4 replicative form replication takes place by a displacement mechanism in which synthesis of the new viral DNA strand displaces the old viral DNA strand as a single-stranded DNA loop (D-loop) and when the displacement reaches half way round the molecule (the origin of synthesis of the G4 viral and complementary DNA strands are on opposite sides of the genome, Martin &; Godson 1977) synthesis of the complementary DNA strand starts, but in the opposite direction. Strand separation of the parent helix runs ahead of DNA synthesis, releasing two partially single-stranded circles from the replicating structure which then complete their replication as free single-stranded DNA circles. No evidence was found to support a rolling circle displacement mechanism of G4 replicative form synthesis. 相似文献
6.
S Eisenberg 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1980,210(1180):337-349
The gene A protein of bacteriophage phi X 174 initiates replication of super-twisted RFI DNA by cleaving the viral (+) strand at the origin of replication and binding to the 5' end. Upon addition of E. coli rep protein (single-stranded DNA dependent ATPase), E. coli single-stranded DNA binding protein and ATP, complete unwinding of the two strands occurs. Electron microscopic analyses of intermediates in the reaction reveal that the unwinding occurs by movement of the 5' end into the duplex, displacing the viral strand in the form of a single-stranded loop. Since unwinding will not occur in the absence of either gene A protein or rep protein, it is presumed that the rep protein interacts to form a complex with the bound gene A protein. Single-stranded DNA binding protein facilitates the unwinding by binding to the exposed single-stranded DNA. Further addition of the four deoxyribotriphosphates and DNA polymerase III holoenzyme to the reaction results in synthesis of viral (+) single-stranded circles in amounts exceeding that of the input template. A model describing the role of gene A protein and rep protein in duplex DNA replication is presented and other properties of gene A protein discussed. 相似文献
7.
Drosophila melanogaster strand transferase. A protein that forms heteroduplex DNA in the absence of both ATP and single-strand DNA binding protein 总被引:16,自引:0,他引:16
The purification of a Drosophila strand transfer protein is described, which involves Bio-Rex 70, Superose 6, Mono S, and single-stranded DNA-agarose chromatography. A 105,000-dalton polypeptide copurifies with the strand transfer activity on the last two column steps. The strand transferase carries out strand transfer at an unusually low protein:single-stranded DNA ratio and requires neither a nucleotide cofactor nor exogenous single-strand DNA binding protein to form heteroduplex DNA. Biochemical analysis of the reaction products has established that one strand of the DNA duplex is displaced during the reaction. Several properties, including the kinetics and stoichiometry of strand transfer, differentiate this activity from previously characterized strand transferases. 相似文献
8.
Purified Rad3 protein from the yeast Saccharomyces cerevisiae is a single-stranded DNA-dependent ATPase and also acts as a DNA helicase on partially duplex DNA. In this study we show that the DNA helicase activity is inhibited when a partially duplex circular DNA substrate is exposed to ultraviolet (UV) radiation. Inhibition of DNA helicase activity is sensitive to the particular strand of the duplex region which carries the damage. Inhibition is retained if the single-stranded circle is irradiated prior to annealing to an unirradiated oligonucleotide, but not if a UV-irradiated oligonucleotide is annealed to unirradiated circular single-stranded DNA. UV irradiation of single-stranded DNA or deoxyribonucleotide homopolymers also inhibits the ability of these polynucleotides to support the hydrolysis of ATP by Rad3 protein. UV radiation damage apparently blocks translocation of Rad3 protein and results in the formation of stable Rad3 protein-UV-irradiated DNA complexes. As a consequence, Rad3 protein remains sequestered on DNA, presumably at sites of base damage. The sensitivity of Rad3 protein to the presence of DNA damage on the strand along which it translocates provides a potential mechanism for damage recognition during nucleotide excision repair and may explain the absolute requirement for Rad3 protein for damage-specific incision of DNA in yeast. 相似文献
9.
Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange. 总被引:7,自引:1,他引:6
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Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein. 相似文献
10.
Gene II protein is required for all phases of filamentous phage DNA synthesis other than the conversion of the infecting single strand to the parental double-stranded molecule. It introduces a specific nick into the double-stranded replicative form DNA, is required for the initiation of (+) strand synthesis and is responsible for termination and ring closure of the (+) strand product. Here we show that the gene II protein also promotes minus strand synthesis later in infection. Over-expression of gene II protein can induce the conversion of all nascent single-stranded phage DNA to the double-stranded form, even in the presence of the single-stranded DNA-binding gene V protein that would normally sequester the newly synthesized single strands. We also present evidence that the gene X protein (separately translated from an initiator codon within gene II, and identical to the C-terminal one-third of the gene II protein) is a powerful inhibitor of phage-specific DNA synthesis in vivo. 相似文献
11.
A proteolytic fragment of recA protein, missing about 15% of the protein at the C terminus, was found to promote assimilation of homologous single-stranded DNA into duplex DNA more efficiently than intact recA protein. This difference was not found if Escherichia coli single-stranded DNA binding protein was present. The ATPase activity of both intact recA protein and the fragment was identical. The difference in strand assimilation activity cannot be due to differences in single-stranded DNA affinity, since both the fragment and intact proteins bind to single-stranded DNA with nearly identical affinities. However, the fragment was found to bind double-stranded DNA more tightly and to aggregate more extensively than recA protein; both of these properties may be important in strand assimilation. Aggregation of the fragment was extensive in the presence of duplex DNA under the same condition where recA protein did not aggregate. The double-stranded DNA binding of both recA protein and the fragment responds to nucleotide cofactors in the same manner as single-stranded DNA binding, i.e. ADP weakens and ATP gamma S strengthens the association. The missing C-terminal region of recA protein includes a very acidic region that is homologous to other single-stranded DNA binding proteins and which has been implicated in DNA binding modulation. This C-terminal region may serve a similar function in recA protein, possibly inhibiting double-stranded DNA invasion. The possible role of the enhanced double-stranded DNA affinity of the fragment protein in the mechanism of strand assimilation is discussed. 相似文献
12.
Bacteriophage P2 DNA replication. Characterization of the requirement of the gene B protein in vivo 总被引:3,自引:0,他引:3
Replicative intermediates isolated from Escherichia coli cells infected with P2 gene B mutants were circular DNA molecules with single-stranded DNA tails, as opposed to the double-stranded DNA tails of wild-type replicative intermediates. The results show that the mutant replicative intermediates arose from aberrant DNA replication, aberrant due to a lack of lagging strand DNA synthesis, but with normal leading strand synthesis, so that only one circular duplex daughter DNA molecule was made from each duplex parent molecule. The single-stranded tails were shown to correspond to the nicked (and therefore displaced) parental DNA "l" strands. By partial denaturation mapping, the ends of the single-stranded tails tended to map close to the replication origin, but not all at a unique position, probably due to partial degradation or breakage in vivo, or during cell lysis or DNA isolation. By hybridization to separated strands of P2 DNA on nitrocellulose filters, DNA synthesis was shown to be asymmetric, and consistent with more leading strand than lagging strand synthesis having occurred. We concluded that the gene B protein is required for lagging strand DNA synthesis, but not for initiation, elongation or termination of the leading strand. 相似文献
13.
In this issue of Structure, He and colleagues present the crystal structure of the phage T4 Dda helicase bound to DNA, providing new insights into how this protein efficiently couples single-stranded DNA translocation to DNA strand separation. 相似文献
14.
ATP utilization by rep protein in the catalytic separation of DNA strands at a replicating fork. 总被引:13,自引:0,他引:13
Hydrolysis of ATP by rep protein proceeds in the presence of a single-stranded region of DNA 4 residues long, but the true effector for rep ATPase appears to be a replicating fork rather than a random coil. At or near a fork in duplex DNA, rep ATPase action is different from what it is on DNA lacking secondary structure (single-stranded): (i) Km for ATP is lower, (ii) specificity is for ATP and dATP with no action on other nucleoside triphosphates, (iii) sensitivity to certain ATP analogs is reduced, (iv) presence of a DNA-nicking enzyme (e.g. cistron A protein induced by phiX174) is required, and (v) Escherichia coli DNA binding protein facilitates rather than inhibits. During the separation of strands accompanying replication, 2 molecules of nucleoside triphosphate (ATP or dATP) are hydrolyzed for every nucleotide polymerized. Utilization of ATP by rep protein may provide energy for catalytic strand separation at a fork in advance of replication. 相似文献
15.
In the bacteriophage T4 DNA replication system, T4 gene 59 protein binds preferentially to fork DNA and accelerates the loading of the T4 41 helicase. 59 protein also binds the T4 32 single-stranded DNA-binding protein that coats the lagging strand template. Here we explore the function of the strong affinity between the 32 and 59 proteins at the replication fork. We show that, in contrast to the 59 helicase loader, 32 protein does not bind forked DNA more tightly than linear DNA. 32 protein displays a strong binding polarity on fork DNA, binding with much higher affinity to the 5' single-stranded lagging strand template arm of a model fork, than to the 3' single-stranded leading strand arm. 59 protein promotes the binding of 32 protein on forks too short for cooperative binding by 32 protein. We show that 32 protein is required for helicase-dependent leading strand DNA synthesis when the helicase is loaded by 59 protein. However, 32 protein is not required for leading strand synthesis when helicase is loaded, less efficiently, without 59 protein. Leading strand synthesis by wild type T4 polymerase is strongly inhibited when 59 protein is present without 32 protein. Because 59 protein can load the helicase on forks without 32 protein, our results are best explained by a model in which 59 helicase loader at the fork prevents the coupling of the leading strand polymerase and the helicase, unless the position of 59 protein is shifted by its association with 32 protein. 相似文献
16.
The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer 总被引:11,自引:4,他引:7
The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer ( oriT ). The absence of MobC has several different effects on oriT DNA. Site- and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA. 相似文献
17.
Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis 总被引:13,自引:12,他引:1
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Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings. 相似文献
18.
Selmane T Wittung-Stafshede P Maraboeuf F Voloshin ON Nordén B Camerini-Otero DR Takahashi M 《FEBS letters》1999,446(1):30-34
The L2 loop in the RecA protein is the catalytic center for DNA strand exchange. Here we investigate the DNA binding properties of the L2 loop peptide using optical spectroscopy with polarized light. Both fluorescence intensity and anisotropy of an etheno-modified poly(dA) increase upon peptide binding, indicate that the base motions of single-stranded DNA are restricted in the complex. In agreement with this conclusion, the peptide-poly(dT) complex exhibits a significant linear dichroism signal. The peptide is also found to modify the structure of double-stranded DNA, but does not denature it. It is inferred that strand separation may not be required for the formation of a joint molecule. 相似文献
19.
The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. 总被引:4,自引:1,他引:3
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RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site. 相似文献
20.
During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state. 相似文献