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1.
Primer-terminus stabilization at the 3'-5' exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases. 总被引:2,自引:0,他引:2
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By site-directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3'-5' exonuclease domain of DNA-dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single-stranded DNA (ssDNA) ligands at the 3'-5' exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3'-5' exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3'-5' exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA-dependent DNA polymerases. Moreover, in contrast to the dual role in 3'-5' exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer-terminus at the 3'-5' exonuclease active site. 相似文献
2.
3.
Kuroita T Matsumura H Yokota N Kitabayashi M Hashimoto H Inoue T Imanaka T Kai Y 《Journal of molecular biology》2005,351(2):291-298
A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases. 相似文献
4.
Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity. 总被引:4,自引:0,他引:4
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The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA. 相似文献
5.
Blanca G Delagoutte E Tanguy le Gac N Johnson NP Baldacci G Villani G 《The Biochemical journal》2007,402(2):321-329
Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3'-5' exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3'-5' exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo- polymerase (T4 DNA polymerase deficient in the 3'-5' exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo- polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis. 相似文献
6.
Nishioka M Mizuguchi H Fujiwara S Komatsubara S Kitabayashi M Uemura H Takagi M Imanaka T 《Journal of biotechnology》2001,88(2):141-149
DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus sp. KOD1) is one of the most efficient thermostable PCR enzymes exhibiting higher accuracy and elongation velocity than any other commercially available DNA polymerase [M. Takagi et al. (1997) Appl. Environ. Microbiol. 63, 4504-4510]. However, when long distance PCR (>5 kbp) was performed with KOD DNA polymerase, amplification efficiency (product yield) becomes lower because of its strong 3'-5' exonuclease activity for proof-reading. In order to improve a target length limitation in PCR, mutant DNA polymerases with decreased 3'-5' exonuclease activity were designed by substituting amino acid residues in conserved exonuclease motifs, Exo I (Asp141-Xaa-Glu), Exo II (Asn210-Xaa-Xaa-Xaa-Phe-Asp), and Exo III (Tyr311-Xaa-Xaa-Xaa-Asp). Exonuclease activity and amplification fidelity (error rate) of the DNA polymerases were altered by mutagenesis. However, long and accurate PCR by a single-type of mutant DNA polymerase was very difficult. The wild-type DNA polymerase (WT) and its exonuclease deficient mutant (N210D) were mixed in different ratio and their characteristics in PCR were examined. When the mixed enzyme (WT and N210D) was made at the ratio of 1:40, long PCR (15 kbp) at lower mutation frequency could be efficiently achieved. 相似文献
7.
Polymerization activity of an alpha-like DNA polymerase requires a conserved 3''-5'' exonuclease active site. 总被引:9,自引:0,他引:9
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J S Gibbs K Weisshart P Digard A deBruynKops D M Knipe D M Coen 《Molecular and cellular biology》1991,11(9):4786-4795
Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases. 相似文献
8.
The intervening domain of the thermostable Thermus aquaticus DNA polymerase (TAQ: polymerase), which has no catalytic activity, has been exchanged for the 3'-5' exonuclease domain of the homologous mesophile Escherichia coli DNA polymerase I (E.coli pol I) and the homologous thermostable Thermotoga neapolitana DNA polymerase (TNE: polymerase). Three chimeric DNA polymerases have been constructed using the three-dimensional (3D) structure of the Klenow fragment of the E.coli pol I and 3D models of the intervening and polymerase domains of the TAQ: polymerase and the TNE: polymerase: chimera TaqEc1 (exchange of residues 292-423 from TAQ: polymerase for residues 327-519 of E.coli pol I), chimera TaqTne1 (exchange of residues 292-423 of TAQ: polymerase for residues 295-485 of TNE: polymerase) and chimera TaqTne2 (exchange of residues 292-448 of TAQ: polymerase for residues 295-510 of TNE: polymerase). The chimera TaqEc1 showed characteristics from both parental polymerases at an intermediate temperature of 50 degrees C: high polymerase activity, processivity, 3'-5' exonuclease activity and proof-reading function. In comparison, the chimeras TaqTne1 and TaqTne2 showed no significant 3'-5' exonuclease activity and no proof-reading function. The chimera TaqTne1 showed an optimum temperature at 60 degrees C, decreased polymerase activity compared with the TAQ: polymerase and reduced processivity. The chimera TaqTne2 showed high polymerase activity at 72 degrees C, processivity and less reduced thermostability compared with the chimera TaqTne1. 相似文献
9.
The Klenow fragment of Escherichia coli DNA polymerase I houses catalytic centers for both polymerase and 3'-5' exonuclease activities that are separated by about 35 A. Upon the incorporation of a mismatched nucleotide, the primer terminus is transferred from the polymerase site to an exonuclease site designed for excision of the mismatched nucleotides. The structural comparison of the binary complexes of DNA polymerases in the polymerase and the exonuclease modes, together with a molecular modeling of the template strand overhang in Klenow fragment, indicated its binding in the region spanning residues 821-824. Since these residues are conserved in the "A" family DNA polymerases, we have designated this region as the RRRY motif. The alanine substitution of individual amino acid residues of this motif did not change the polymerase activity; however, the 3'-5' exonuclease activity was reduced 2-29-fold, depending upon the site of mutation. The R821A and R822A/Y824A mutant enzymes showed maximum cleavage defect with single-stranded DNA, mainly due to a large decrease in the ssDNA binding affinity of these enzymes. Mismatch removal by these enzymes was only moderately affected. However, data from the exonuclease-polymerase balance assays with mismatched template-primer suggest that the mutant enzymes are defective in switching mismatched primer from the polymerase to the exonuclease site. Thus, the RRRY motif provides a binding track for substrate ssDNA and for nonsubstrate single-stranded template overhang, in a polarity-dependent manner. This binding then facilitates cleavage of the substrate at the exonuclease site. 相似文献
10.
Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I 总被引:1,自引:0,他引:1
The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II). 相似文献
11.
12.
On the fidelity of DNA replication: herpes DNA polymerase and its associated exonuclease. 总被引:1,自引:0,他引:1
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Procaryotic DNA polymerases contain an associated 3'----5' exonuclease activity which provides a proofreading function and contributes substantially to replication fidelity. DNA polymerases of the eucaryotic herpes-type viruses contain similar associated exonuclease activities. We have investigated the fidelity of polymerases purified from wild type herpes simplex virus, as well as from mutator and antimutator strains. On synthetic templates, the herpes enzymes show greater relative exonuclease activities, and greater ability to excise a terminal mismatched base, than procaryotic DNA polymerases which proofread. On a phi X174 natural DNA template, the herpes enzymes are more accurate than purified eucaryotic DNA polymerases; the error rate is similar to E. coli polymerase I. However, conditions which abnegate proofreading by E. coli polymerase I have little effect on the herpes enzymes. We conclude that either these viral polymerases are accurate in the absence of proofreading, or the conditions examined have little effect on proofreading by the herpes DNA polymerases. 相似文献
13.
E Elisseeva S S Mandal L J Reha-Krantz 《The Journal of biological chemistry》1999,274(35):25151-25158
The 3' --> 5' exonuclease activity of proofreading DNA polymerases requires two divalent metal ions, metal ions A and B. Mutational studies of the 3' --> 5' exonuclease active center of the bacteriophage T4 DNA polymerase indicate that residue Asp-324, which binds metal ion A, is the single most important residue for the hydrolysis reaction. In the absence of a nonenzymatic source of hydroxide ions, an alanine substitution for residue Asp-324 reduced exonuclease activity 10-100-fold more than alanine substitutions for the other metal-binding residues, Asp-112 and Asp-219. Thus, exonuclease activity is reduced 10(5)-fold for the D324A-DNA polymerase compared with the wild-type enzyme, while decreases of 10(3)- to 10(4)-fold are detected for the D219A- and D112A/E114A-DNA polymerases, respectively. Our results are consistent with the proposal that a water molecule, coordinated by metal ion A, forms a metal-hydroxide ion that is oriented to attack the phosphodiester bond at the site of cleavage. Residues Glu-114 and Lys-299 may assist the reaction by lowering the pK(a) of the metal ion-A coordinated water molecule, whereas residue Tyr-320 may help to reorient the DNA from the binding conformation to the catalytically active conformation. 相似文献
14.
Single-base discrimination mediated by proofreading 3' phosphorothioate-modified primers 总被引:3,自引:0,他引:3
It has been well known for decades that deoxyribonucleic acid (DNA) polymerases with proofreading function have a higher fidelity in primer extension as compared to those without 3' exonuclease activities. However, polymerases with proofreading function have not been used in single nucleotide polymorphism (SNP) assays. Here, we describe a new method for single-base discrimination by proofreading the 3' phosphorothioate-modified primers using a polymerase with proofreading function. Our data show that the combination of a polymerase with 3' exonuclease activity and the 3' phosphorothioate-modified primers work efficiently as a single-base mismatch-operated on/off switch. DNA polymerization only occurred from matched primers, whereas mismatched primers were not extended at the broad range of annealing temperature tested in our study. This novel single-base discrimination method has potential in SNP assays. 相似文献
15.
DNA polymerases from the A and B families with 3'-5' exonucleolytic activity have exonuclease domains with similar three-dimensional structures that require two divalent metal ions for catalysis. B family DNA polymerases that are part of a replicase generally have a more potent 3'-5' exonuclease (exo) activity than A family DNA polymerases that mainly function in DNA repair. To investigate the basis for these differences, we determined pH-activity profiles for the exonuclease reactions of T4, RB69, and phi29 DNA polymerases as representatives of B family replicative DNA polymerases and the Klenow fragment (KF) as an example of a repair DNA polymerase in the A family. We performed exo assays under single-turnover conditions and found that excision rates exhibited by the B family DNA polymerases were essentially independent of pH between pH 6.5 and 8.5, whereas the exo activity of KF increased 10-fold for each unit increase in pH. Three exo domain mutants of RB69 polymerase had much lower exo activities than the wild-type enzyme and exhibited pH-activity profiles similar to that of KF. On the basis of pH versus activity data and elemental effects obtained using short double-stranded DNA substrates terminating in phosphorothioate linkages, we suggest that the rate of the chemical step is reduced to the point where it becomes limiting with RB69 pol mutants K302A, Y323F, and E116A, in contrast to the wild-type enzyme where chemistry is faster than the rate-determining step that precedes it. 相似文献
16.
M Bakhanashvili 《European journal of biochemistry》2001,268(7):2047-2054
17.
Shen Y Musti K Hiramoto M Kikuchi H Kawarabayashi Y Matsui I 《The Journal of biological chemistry》2001,276(29):27376-27383
Family D DNA polymerase has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair, and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. Although it possesses strong polymerization and proofreading activities, the motifs common to other DNA polymerase families are absent in its sequences. Here we report the mapping of the catalytic residues in a family D DNA polymerase from Pyrococcus horikoshii. Site-directed alanine mutants for 28 conserved aspartic acid or glutamic acid residues were screened for polymerization and 3'-5' exonuclease activities. We identified the invariant aspartates Asp-1122 and Asp-1124 within the most conserved motif as the catalytic residues involved in DNA polymerization. Alanine mutation at either site caused a loss of polymerization activity, whereas the conserved mutants, D1122E, D1124N, and D1124E, had slightly reduced polymerization activity. We also found that the 3'-5' exonuclease activity remains in D1122A and D1124A, indicating that the catalytic residues of DNA polymerization are different from those of the 3'-5' exonuclease activity. Furthermore we determined the molecular mass of the recombinant enzyme by gel filtration and proposed a heterotetrameric structure for this enzyme. 相似文献
18.
Shimizu K Hashimoto K Kirchner JM Nakai W Nishikawa H Resnick MA Sugino A 《The Journal of biological chemistry》2002,277(40):37422-37429
DNA polymerases delta and epsilon (pol delta and epsilon) are the major replicative polymerases and possess 3'-5' proofreading exonuclease activities that correct errors arising during DNA replication in the yeast Saccharomyces cerevisiae. This study measures the fidelity of the holoenzyme of wild-type pol epsilon, the 3'-5' exonuclease-deficient pol2-4, a +1 frameshift mutator for homonucleotide runs, pol2C1089Y, and pol2C1089Y pol2-4 enzymes using a synthetic 30-mer primer/100-mer template. The nucleotide substitution rate for wild-type pol epsilon was 0.47 x 10(-5) for G:G mismatches, 0.15 x 10(-5) for T:G mismatches, and less than 0.01 x 10(-5) for A:G mismatches. The accuracy for A opposite G was not altered in the exonuclease-deficient pol2-4 pol epsilon; however, G:G and T:G misincorporation rates increased 40- and 73-fold, respectively. The pol2C1089Y pol epsilon mutant also exhibited increased G:G and T:G misincorporation rates, 22- and 10-fold, respectively, whereas A:G misincorporation did not differ from that of wild type. Since the fidelity of the double mutant pol2-4 pol2C1089Y was not greatly decreased, these results suggest that the proofreading 3'-5' exonuclease activity of pol2C1089Y pol epsilon is impaired even though it retains nuclease activity and the mutation is not in the known exonuclease domain. 相似文献
19.
Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase. 相似文献
20.
Purified DNA polymerase alpha, the major replicating enzyme found in mammalian cells, lacks an associated 3'----5' proofreading exonuclease that, in bacteria, contributes significantly to the accuracy of DNA replication. Calf thymus DNA polymerase alpha cannot remove mispaired 3'-termini, nor can it extend them efficiently. We designed a biochemical assay to search in cell extracts for a putative proofreading exonuclease that might function in concert with DNA polymerase alpha in vivo but dissociates from it during purification. Using this assay, we purified a 3'----5' exonuclease from calf thymus that preferentially hydrolyzes mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by DNA polymerase alpha. This exonuclease copurifies with a DNA polymerase activity that is biochemically distinct from DNA polymerase alpha and exhibits characteristics described for a second replicative DNA polymerase, DNA polymerase delta. In related studies, we showed that the 3'----5' exonuclease of authentic DNA polymerase delta, like the purified exonuclease, removes terminal mispairs, allowing extension by DNA polymerase alpha. These data suggest that a single proofreading exonuclease could be shared by DNA polymerases alpha and delta, functioning at the site of DNA replication in mammalian cells. 相似文献