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1.
Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. I. Multiple effectors act to modulate Okazaki fragment size. 总被引:11,自引:0,他引:11
The coordinated action of many enzymatic activities is required at the DNA replication fork to ensure the error-free, efficient, and simultaneous synthesis of the leading and lagging strands of DNA. In order to define the essential protein-protein interactions and model the regulatory pathways that control Okazaki fragment synthesis, we have reconstituted the replication fork of Escherichia coli in vitro in a rolling circle-type DNA replication system. In this system, in the presence of the single-stranded DNA binding protein, the helicase/primase function on the lagging-strand template is provided by the primosome, and the synthesis of DNA strands is catalyzed by the DNA polymerase III holoenzyme. These reconstituted replication forks synthesize equivalent amounts of leading- and lagging-strand DNA, move at rates comparable to those measured in vivo (600-800 nucleotides/s at 30 degrees C), and can synthesize leading strands in the range of 150-500 kilobases in length. Using this system, we have studied the cycle of Okazaki fragment synthesis at the replication fork. This cycle is likely to have several well defined decision points, steps in the cycle where incorrect execution by the enzymatic machinery will result in an alteration in the product of the reaction, i.e. in the size of the Okazaki fragments. Since identification of these decision points should aid in the determination of which of the enzymes acting at the replication fork control the cycle, we have endeavored to identify those reaction parameters that, when varied, alter the size of the Okazaki fragments synthesized. Here we demonstrate that some enzymes, such as the DnaB helicase, remain associated continuously with the fork while others, such as the primase, must be recruited from solution each time synthesis of an Okazaki fragment is initiated. We also show that variation of the concentration of the ribonucleoside triphosphates and the deoxyribonucleoside triphosphates affects Okazaki fragment size, that the control mechanisms acting at the fork to control Okazaki fragment size are not fixed at the time the fork is assembled but can be varied during the lifetime of the fork, and that alteration in the rate of the leading-strand DNA polymerase cannot account for the effect of the deoxyribonucleoside triphosphates. 相似文献
2.
To investigate the role of the priming apparatus at the replication fork in determining Okazaki fragment size, the products of primer synthesis generated in vitro during rolling-circle DNA replication catalyzed by the DNA polymerase III holoenzyme, the single-stranded DNA binding protein, and the primosome on a tailed form II DNA template were isolated and characterized. The abundance of oligoribonucleotide primers and the incidence of covalent DNA chain extension of the primer population was measured under different reaction conditions known to affect the size of the products of lagging-strand DNA synthesis. These analyses demonstrated that the factors affecting Okazaki fragment length could be distinguished by either their effect on the frequency of primer synthesis or by their influence on the efficiency of initiation of DNA synthesis from primer termini. Primase and the ribonucleoside triphosphates were found to stimulate primer synthesis. The observed trend toward smaller fragment size as the concentration of these effectors was raised was apparently a direct consequence of the increased frequency of primer synthesis. The beta subunit of the DNA polymerase III holoenzyme and the deoxyribonucleoside triphosphates did not alter the priming frequency; instead, the concentration of these factors influenced the ability of the lagging-strand DNA polymerase to efficiently utilize primers to initiate DNA synthesis. Maximum utilization of the available primers correlated with the lowest mean value of Okazaki fragment length. These data were used to draw general conclusions concerning the temporal order of enzymatic steps that operate during a cycle of Okazaki fragment synthesis on the lagging-strand DNA template. 相似文献
3.
Studies with a rolling-circle DNA replication system reconstituted in vitro with a tailed form II DNA template, the DNA polymerase III holoenzyme (Pol III HE), the Escherichia coli single-stranded DNA binding protein, and the primosome, showed that within the context of a replication fork, the oligoribonucleotide primers that were formed were limited to a length in the range of 9 to 14 nucleotides, regardless of whether they were subsequently elongated by the lagging-strand DNA polymerase. This is in contrast to the 8-60-nucleotide-long primers synthesized by the primosome in the absence of DNA replication on a bacteriophage phi X174 DNA template, although when primer synthesis and DNA replication were catalyzed concurrently in this system, the extent of RNA polymerization decreased. As described in this report, we therefore examined the effect of the DNA Pol III HE on the length of primers synthesized by primase in vitro in the absence of DNA replication. When primer synthesis was catalyzed either: i) by the primosome on a phi X174 DNA template, ii) by primase on naked DNA with the aid of the DnaB protein (general priming), or iii) by primase alone at the bacteriophage G4 origin, the presence of the DNA Pol III HE in the reaction mixtures resulted in a universal reduction in the length of the heterogeneous RNA products to a uniform size of approximately 10 nucleotides. dNTPs were not required, and the addition of dGMP, an inhibitor of the 3'----5' exonuclease of the DNA Pol III HE, did not alter the effect; therefore, neither the 5'----3' DNA polymerase activity nor the 3'----5' exonuclease activity of the DNA Pol III HE was involved. E. coli DNA polymerase I, and the DNA polymerases of bacteriophages T4 and T7 could not substitute for the DNA Pol III HE. The Pol III core plays a crucial role in mediating this effect, although other subunits of the DNA Pol III HE are also required. These observations suggest that the association of primase with the DNA Pol III HE during primer synthesis regulates its catalytic activity and that this regulatory interaction occurs independently of, and prior to, formation of a preinitiation complex of the DNA Pol III HE on the primer terminus. 相似文献
4.
C A Wu E L Zechner A J Hughes M A Franden C S McHenry K J Marians 《The Journal of biological chemistry》1992,267(6):4064-4073
Individually purified subunits have been used to reconstitute the action of the Escherichia coli DNA polymerase III holoenzyme (Pol III HE) at a replication fork formed in the presence of the primosome, the single-stranded DNA binding protein, and a tailed form II DNA template. Complete activity, indistinguishable from that of the intact DNA Pol III HE, could be reproduced with a combination of the DNA polymerase III core (Pol III core), the gamma.delta complex, and the beta subunit. Experiments where the Pol III core in reaction mixtures containing active replication forks was diluted suggested that the lagging-strand Pol III core remained associated continuously with the replication fork through multiple cycles of Okazaki fragment synthesis. Since the lagging-strand Pol III core must dissociate from the 3' end of the completed Okazaki fragment, this suggests that its association with the fork is via protein-protein interactions, lending credence to the idea that it forms a dimeric complex with the leading-strand Pol III core. An asymmetry in the action of the subunits was revealed under conditions (high ionic strength) that were presumably destabilizing to the integrity of the replication fork. Under these conditions, tau acted to stimulate DNA synthesis only when the primase was present (i.e. when lagging-strand DNA synthesis was ongoing). This stimulation was reflected by an inhibition of the formation of small Okazaki fragments, suggesting that, within the context of the model developed to account for the temporal order of steps during a cycle of Okazaki fragment synthesis, the presence of tau accelerated the transit of the lagging-strand Pol III core from the 3' end of the completed Okazaki fragment to the 3' end of the new primer. 相似文献
5.
Analysis of mammalian mtDNA by two-dimensional agarose gel electrophoresis revealed two classes of replication intermediate. One was resistant to single-strand nuclease digestion and displayed the mobility properties of coupled leading- and lagging- strand replication products. Intermediates of coupled, unidirectional mtDNA replication were found in mouse liver and human placenta and were the predominant species in cultured cells recovering from transient mtDNA replication. Replication intermediates sensitive to single-strand nuclease were most abundant in untreated cultured cells. These are presumed to derive from the orthodox, strand-asynchronous mode of mtDNA replication. These findings indicate that two modes of mtDNA replication operate in mammalian cells and that changes in mtDNA copy number involve an alteration in the mode of mtDNA replication. 相似文献
6.
We have demonstrated previously that the template sequences 5'-GTT-3' and 5'-GCT-3' serve as necessary and sufficient signals for the initiation of new DNA chains that start with pentaribonucleotide primers of sequence pppApCpNpNpN or pppGpCpNpNpN, respectively. Normally, the complete T4 primosome, consisting of the T4 gene 41 (DNA helicase) and gene 61 (primase) proteins, is required to produce RNA primers. However, a high concentration of the 61 protein alone can prime DNA chain starts from the GCT sites [Cha, T.-A., & Alberts, B. M. (1986) J. Biol. Chem. 261, 7001-7010]. We show here that the 61 protein can catalyze a single-stranded DNA template-dependent reaction in which the dimers pppApC and pppGpC are the major products and much longer oligomers of various lengths are minor ones. Further addition of the 41 protein is needed to form a primosome that catalyzes efficient synthesis of the physiologically relevant pentaribonucleotides that are responsible for the de novo DNA chain starts on the lagging strand of a replication fork. The helicase activity of the 41 protein is necessary and sufficient to ensure a high rate and processivity of DNA synthesis on the leading strand [Cha, T.-A., & Alberts, B. M. (1989) J. Biol. Chem. 264, 12220-12225]. Coupling an RNA primase to this helicase in the primosome therefore coordinates the leading- and lagging-strand DNA syntheses at a DNA replication fork. Our experiments reveal that the addition of the T4 helix-destabilizing protein (the gene 32 protein) is required to confine the synthesis of RNA primers to those sites where they are used to start an Okazaki fragment, causing many potential priming sites to be passed by the primosome without triggering primer synthesis. 相似文献
7.
We present results which suggest considerable flexibility in the RNA priming of Okazaki pieces at the E. coli replication fork. Using film lysates on cellophane discs, we have identified RNA at the 5' ends of Okazaki pieces. All four ribonucleotides are found to be present at the RNA-DNA junction if all four ribonucleoside triphosphates are used. However, if only ATP, or ATP and GTP are used, then only 2' (3')AMP, or 2' (3')AMP and 2' (3')GMP are found at the RNA-DNA junction. A nearest neighbor analysis of RNA associated with Okazaki pieces using alpha 32P-CTP as a probe shows a similar dependence of nearest neighbor composition on the ribonucleoside triphosphate composition of the incubation mixture. Thus, the nucleotide composition of the RNA primers at the ends of Okazaki pieces varies as a function of the ribonucleoside triphosphates available. 相似文献
8.
Okazaki pieces grow opposite to the replication fork direction during simian virus 40 DNA replication. 总被引:15,自引:4,他引:11
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Simian virus 40 replicating DNA was pulse labeled with alpha-32P-dATP using an acellular DNA replication system. Nascent DNA chains of less than 200 nucleotides (Okazaki pieces) were then isolated from the denatured replicating DNA by electrosieving through a polyacrylamide gel column. The purified Okazaki pieces were hybridized to separated strands of Bg1(1)+Hpa1 simian virus 40 DNA restriction fragments immobilized on nitrocellulose filters. Only strands with polarity of the DNA replication fork direction hybridized with Okazaki pieces. Hence, Okazaki pieces in simian virus 40 are synthesized against the DNA replication fork direction. 相似文献
9.
We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome. 相似文献
10.
Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell. 相似文献
11.
B M Olivera P Manlapaz-Ramos H R Warner B K Duncan 《Journal of molecular biology》1979,128(3):265-275
The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung).Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool).These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung+ strain.A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung+ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut+ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue. 相似文献
12.
The initiation of the DNA replication cycle in Escherichia coli requires protein synthesis. Marunouchi &; Messer (1973) have hypothesized that an additional protein synthesis step is required for the replication of the terminal segment of the chromosome, and that replication of this segment is a prerequisite for subsequent cell division. We have not confirmed the existence of a unique terminal segment using a protocol designed to label the hypothesized segment with [3H]dThd2. Our protocol avoids the increased incorporation of [3H]dThd into DNA caused by abrupt increases in temperature, a complication implicit in the technique of Marunouchi &; Messer (1973).Treatment with nalidixic acid (an inhibitor of semiconservative DNA synthesis) in sufficient concentration to prevent replication of the postulated terminal segment prevents cell division but also causes loss of viability. This makes it difficult to correlate the effect of nalidixic acid on cell division with DNA synthesis inhibition alone. 相似文献
13.
14.
A mutant of Escherichia coli (sof) which was previously shown to have increased recombination frequency, to produce abnormally short "Okazaki fragments," and to be deficient in deoxyuridine triphosphatase has now been found also to possess mutator activity for several genes; point mutation rates and deletion rates are affected. The mutational stimulation effects are consistent with the hypothesis that incorporation of uracil into DNA is directly or indirectly responsible for the observed mutator activity. 相似文献
15.
Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner. 相似文献
16.
Gawel D Jonczyk P Bialoskorska M Schaaper RM Fijalkowska IJ 《Mutation research》2002,501(1-2):129-136
Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication. 相似文献
17.
Blockage of the progress of a DNA replication fork in Escherichia coli can be ascribed to an inhibition of helicase action at the orientation-specific binding of a termination sequence (ter) by the ter-binding protein (Lee, E.H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9104-9108). These observations have been extended to include the PriA helicase, thus confirming that blockage is general for helicases. The site of arrest of synthesis by a replication fork is at the very first nucleotide of the 22-base pair E. coli-terB sequence. Strand displacement by DNA polymerases is also inhibited, but is less profound and is orientation-specific. The ter sequences of plasmids R1-terR and -terL and of plasmids R6K and R100 have been compared with those of E. coli-terA and -terB. 相似文献
18.
Herpes simplex virus DNA synthesis at a preformed replication fork in vitro. 总被引:6,自引:4,他引:2
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Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork. 相似文献
19.
A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin. 相似文献
20.
Using a pair of plasmids carrying the rpsL target sequence in different orientations to the replication origin, we analyzed a large number of forward mutations generated in wild-type and mismatch-repair deficient (MMR(-)) Escherichia coli cells to assess the effects of directionality of replication-fork movement on spontaneous mutagenesis and the generation of replication error. All classes of the mutations found in wild-type cells but not MMR(-) cells were strongly affected by the directionality of replication fork movement. It also appeared that the directionality of replication-fork movement governs the directionality of sequence substitution mutagenesis, which occurred in wild-type cells at a frequency comparable to base substitutions and single-base frameshift mutations. A very strong orientation-dependent hot-spot site for single-base frameshift mutations was discovered and demonstrated to be caused by the same process involved in sequence substitution mutagenesis. It is surprising that dnaE173, a potent mutator mutation specific for sequence substitution as well as single-base frameshift, did not enhance the frequency of the hot-spot frameshift mutation. Furthermore, the frequency of the hot-spot frameshift mutation was unchanged in the MMR(-) strain, whereas the mutHLS-dependent mismatch repair system efficiently suppressed the generation of single-base frameshift mutations. These results suggested that the hot-spot frameshift mutagenesis might be initiated at a particular location containing a DNA lesion, and thereby produce a premutagenic replication intermediate resistant to MMR. Significant numbers of spontaneous single-base frameshift mutations are probably caused by similar mechanisms. 相似文献