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1.
DNA replication normally occurs with high fidelity, but certain "slippery" regions of DNA with tracts of mono-, di-, and trinucleotide repeats are frequently mutation hot spots. We have developed an in vitro assay to study the mechanism of dinucleotide repeat expansion. The primer-template resembles a base excision repair substrate with a single nucleotide gap centered opposite a tract of nine CA repeats; nonrepeat sequences flank the dinucleotide repeats. DNA polymerases are expected to repair the gap, but further extension is possible if the DNA polymerase can displace the downstream oligonucleotide. We report here that the wild type bacteriophage T4 DNA polymerase carries out gap and strand displacement replication and also catalyzes a dinucleotide expansion reaction. Repeat expansion was not detected for an exonuclease-deficient T4 DNA polymerase or for Escherichia coli DNA polymerase I. The dinucleotide repeat expansion reaction catalyzed by wild type T4 DNA polymerase required a downstream oligonucleotide to "stall" replication and 3' --> 5' exonuclease activity to remove the 3'-nonrepeat sequence adjacent to the repeat tract in the template strand. These results suggest that dinucleotide repeat expansion may be stimulated in vivo during DNA repair or during processing of Okazaki fragments. 相似文献
2.
F. A. J. de Vries Ch. J. H. Swart-Idenburg A. de Waard 《Molecular & general genetics : MGG》1972,117(1):60-71
Summary An amber mutant in the head protein of bacteriophage T4D, amH36 has been induced to revert by a mutator, tsL56 in gene 43 (the structural gene for DNA polymerase, de Waard, Paul and Lehman, 1965) which is known to cause errors in replication. As a consequence the known am base triplet is converted to other triplets which assign certain amino acids. The nature of the replication errors has been analyzed by looking at the insertion of amino acids in a peptide from the head protein of 60 independent am
+ revertants. Of these, 38 had incorporated tyrosine (like spontaneous revertants also do) while in 21 cases glutamine was inserted and in one case glutamic acid. With the help of the codon catalogue it could be shown that the L56 polymerase promotes an A:TG:C transition as well as more than one type of transversion. The single revertant which had incorporated glutamic acid clearly represents an A:TC:G transversion. The other transversions leading to the insertion of tyrosine indicate that a C:G pair has been converted. In this case the degeneracy of the code does not allow to differentiate between the transversion C:GG:C and C:GA:T. These findings and the absence of certain amino acids as permissible substituents are discussed with regard to the specificity of the errors in replication made by L56 polymerase. 相似文献
3.
The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions. 相似文献
4.
S K Das 《Biochemical and biophysical research communications》1977,79(1):247-253
Bacteriophage T5-induced DNA polymerase has an associated 3′→5′ exonuclease activity for which both single-stranded and duplex DNA serve as substrate (1). In this report, we demonstrate that hydrolysis of single-stranded DNA homopolymers (template) is inhibited in the presence of complementary (Watson-Crick sense) oligonucleotides (primer). Almost complete inhibition is observed at a primer/template ratio of ? 0.1. Formation of “H-bonded” primer-template complex seems to be necessary for the inhibition of template hydrolysis because (a) similar amounts of noncomplementary oligonucleotides have no detectable effect on the rate of template hydrolysis, and (b) complementary oligonucleotides lose their inhibitory potential at temperatures where the H-bonded primer-template complex is expected to be unstable. From our data, it appears that the inhibition of template hydrolysis in the presence of primer molecules is due to the preferential binding of the enzyme at the 3′-OH terminus of the primer in the primer-template complex. 相似文献
5.
Summary T7 RNA polymerase is synthesized in vitro, dependent on T7 DNA. The in vitro synthesized T7 polymerase has the characteristic properties: resistance to rifampicin and streptolydigin and the typical template specificity. 相似文献
6.
7.
DNA polymerases achieve high-fidelity DNA replication in part by checking the accuracy of each nucleotide that is incorporated and, if a mistake is made, the incorrect nucleotide is removed before further primer extension takes place. In order to proofread, the primer-end must be separated from the template strand and transferred from the polymerase to the exonuclease active center where the excision reaction takes place; then the trimmed primer-end is returned to the polymerase active center. Thus, proofreading requires polymerase-to-exonuclease and exonuclease-to-polymerase active site switching. We have used a fluorescence assay that uses differences in the fluorescence intensity of 2-aminopurine (2AP) to measure the rates of active site switching for the bacteriophage T4 DNA polymerase. There are three findings: (i) the rate of return of the trimmed primer-end from the exonuclease to the polymerase active center is rapid, >500 s−1; (ii) T4 DNA polymerase can remove two incorrect nucleotides under single turnover conditions, which includes presumed exonuclease-to-polymerase and polymerase-to-exonuclease active site switching steps and (iii) proofreading reactions that initiate in the polymerase active center are not intrinsically processive. 相似文献
8.
9.
Differences in replication of a DNA template containing an ethyl phosphotriester by T4 DNA polymerase and Escherichia coli DNA polymerase I
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A DNA template containing a single ethyl phosphotriester was replicated in vitro by the bacteriophage T4 DNA polymerase and by Escherichia coli DNA polymerase I (DNA pol I). Escherichia coli DNA pol I bypassed the lesion efficiently, but partial inhibition was observed for T4 DNA polymerase. The replication block produced by the ethyl phosphotriester was increased at low dNTP concentrations and for a mutant T4 DNA polymerase with an antimutator phenotype, increased proofreading activity, and reduced ability to bind DNA in the polymerase active center. These observations support a model in which an ethyl phosphotriester impedes primer elongation by T4 DNA polymerase by decreasing formation of the ternary DNA polymerase–DNA–dNTP complex. When primer elongation is not possible, proofreading becomes the favored reaction. Apparent futile cycles of nucleotide incorporation and proofreading, the idling reaction, were observed at the site of the lesion. The replication block was overcome by higher dNTP concentrations. Thus, ethyl phosphotriesters may be tolerated in vivo by the up-regulation of dNTP biosynthesis that occurs during the cellular checkpoint response to blocked DNA replication forks. 相似文献
10.
We present a simple and convenient protocol for the direct sequencing of bacteriophage T4 genomic DNA. The method utilizes the thermostable DNA polymerase from Thermus aquaticus (Taq) and 32P-end-labeled oligodeoxyribonucleotide primers to produce extension products that allow the analysis of at least 200 nucleotides (nt) on a single sequencing gel. Single-nt changes in the template were easily detectable following an overnight exposure of the autoradiograms. Comparison of sequences from fully modified T4 DNA containing glucosylated hydroxymethyldeoxycytosine or from templates containing cytosine showed little difference in sequence clarity. These techniques considerably simplify the molecular analysis of T-even bacteriophages and should be compatible with automated sequencing methods which employ 5'-end-labeled primers. 相似文献
11.
The synthesis of the DNA polymerase of bacteriophage T4 is autogenously regulated. This protein (gp43), the product of gene 43, binds to a segment of its mRNA that overlaps its ribosome binding site, and thereby blocks translation. We have determined the Kd of the gp43-operator interaction to be 1.0 x 10(-9) M. The minimum operator sequence to which gp43 binds consists of 36 nucleotides that include a hairpin (containing a 5 base-pair helix and an 8 nucleotide loop) and a single-stranded segment that contains the Shine-Dalgarno sequence of the ribosome binding site. In the distantly related bacteriophage RB69 there is a remarkable conservation of this hairpin and loop sequence at the ribosome binding site of its DNA polymerase gene. We have constructed phage operator mutants that overproduce gp43 in vivo, yet are unchanged for in vivo replication rates and phage yield. We present data that show that the replicative and autoregulatory functions are mutually exclusive activities of this polymerase, and suggest a model for gp43 synthesis that links autoregulation to replicative demand. 相似文献
12.
Pauses at positions of secondary structure during in vitro replication of single-stranded fd bacteriophage DNA by T4 DNA polymerase 总被引:10,自引:0,他引:10
Since bacteriophage T4 DNA polymerase is unable to use duplex DNA molecules as templates (B. Alberts, J. Barry, M. Brittner, M. Davies, H. Hama-Inaba, C. C. Liu, D. Mace, L. Moran, C. F. Morris, J. Piperno, and N. Sinha, 1977, in Nucleic Acids and Protein Recognition, Vogel, H. J., ed., pp. 31–63, Academic Press, New York), a technique involving synchronous and uniquely primed synthesis of DNA on the single-stranded fd DNA by the T4 DNA polymerase has been developed to probe regions exhibiting secondary structure on this genome. As the polymerase proceeds, the template secondary structure acts as a kinetic barrier to delay the continuous chain extension catalyzed by this enzyme. These kinetic pause sites can be mapped by denaturing agarose gel electrophoresis of replication intermediates and used to generate a secondary structure map. Using this method, we are able to establish a list including at least seven plausible stable helical regions in fd DNA. Two of the most stable secondary structures have been mapped near fd sequence positions 3350 and 5650, respectively. The latter has been reported to be the region where fd DNA replication begins (C. P. Gray, R. Sommer, C. Polke, E. Beck, and H. Schaller, 1978, Proc. Nat. Acad. Sci. USA, 75, 50–53). However, the biological function associated with the former has yet to be investigated. Following a two-state model, we estimate the first-order rate constant for progression through the duplex regions near position 5650 in fd DNA to be about 0.042 min?1 for fd DNA synthesis by the T4 DNA polymerase under our reaction conditions. A 7.5-fold increase in this rate constant is obtained upon the addition of the T4 DNA helix destabilizing protein (i.e., gene 32 protein). The general pattern of our secondary structure map agrees well with a computer search for duplex regions on the fd genome. Both the stability and the size of a stable secondary structure at a particular position on the fd template determine the time that the newly made DNA molecules spend at that site. A structure with a stem of less than 8 base pairs does not interrupt significantly the procession of the T4 DNA polymerase during the process of fd DNA synthesis. 相似文献
13.
G K Lindberg J K Rist T A Kunkel A Sugino L B Rothman-Denes 《The Journal of biological chemistry》1988,263(23):11319-11326
Coliphage N4 replication is independent of most host DNA replication functions except for the 5'----3' exonuclease activity of polA, DNA ligase, DNA gyrase, and ribonucleotide reductase (Guinta, D., Stambouly, J., Falco, S. C., Rist, J. K., and Rothman-Denes, L. B. (1986) Virology 150, 33-44). It is therefore expected that N4 codes for most of the functions required for replication of its genome. In this paper we report the purification of the N4-coded DNA polymerase from N4-infected cell extracts by following its activity on a gapped template and in an in vitro complementation system for N4 DNA replication (Rist, J. K., Pearle, M., Sugino, A., and Rothman-Denes, L. B. (1986) J. Biol. Chem. 261, 10506-10510). The enzyme is composed of one polypeptide, Mr 87,000. It is most active on templates containing short gaps synthesizing DNA with high fidelity in a quasi-processive manner. A strong 3'----5' exonuclease activity is associated with the DNA polymerase polypeptide. No 5'----3' exonuclease or strand-displacing activities were detected. 相似文献
14.
P R Chakraborty P Bandyopadhyay H H Huang U Maitra 《The Journal of biological chemistry》1974,249(21):6901-6909
15.
Changes in the primary structure of a mutationally altered ribosomal protein S4 of Escherichia coli 总被引:3,自引:0,他引:3
Summary Protein S4-su6 is a mutationally altered 30 S protein from Escherichia coli. S4-su6 suppresses the expression of streptomycin dependence controlled by another 30S protein. In addition, S4-su6 binds less tightly to its RNA binding site than the wild type form of S4. The present data show that at least seven amino acid replacements have occured in S4-su6. Moreover, the tryptic peptide pattern of S4-su6 is different from that of S4. Therefore, the pleiotropic effects of the mutation responsible for the altered structure of S4-su6 can be accounted for by extensive changes in the primary structure of S4-su6.Supported by the Deutsche Forschungsgemeinschaft as well as the University of Uppsala. 相似文献
16.
Control of bacteriophage T4 DNA polymerase synthesis 总被引:13,自引:0,他引:13
M Russel 《Journal of molecular biology》1973,79(1):83-94
Analysis of sodium dodecyl sulphate/acrylamide gels of 14C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43+ infections or increased synthesis in gene 43? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal. 相似文献
17.
18.
Hideo Ikeda 《Molecular & general genetics : MGG》1986,202(3):518-520
Summary Illegitimate recombination dependent on T4 DNA topoisomerase in a cell-free system has recently been described. In that work, recombinants between two phage DNA molecules were produced by the topoisomerase alone, without an Escherichia coli extract. In this paper, it is shown that recombination between phage and circular plasmid DNA molecules can also be detected in the presence or absence of an E. coli extract but at frequencies two or three orders of magnitude lower than that observed in the phage-phage cross. The frequency is probably lower because multiple recombination is required in the case of the phage-plasmid cross. 相似文献
19.
A method for purifying T4 DNA polymerase from cells harboring overexpression plasmids is described. T4 DNA polymerase is precipitated from induced, lysed cells with polyethyleneimine, then extracted and fractionated further with (NH4)2SO4 before chromatography on a column of single-stranded DNA cellulose. This procedure can be completed in three days and consistently provides enzyme preparations which are at least 98% pure. When necessary, one further chromatography step provides T4 DNA polymerase suitable for recombinant DNA applications. 相似文献