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1.
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
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Vakharia-Rao H Kastead KA Savenkova MI Bulathsinghala CM Postle K 《Journal of bacteriology》2007,189(13):4662-4670
The active transport of iron siderophores and vitamin B(12) across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBDelta7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBDelta7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter. 相似文献
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In the family-B DNA polymerase of bacteriophage RB69, the conserved aromatic palm-subdomain residues Tyr391 and Tyr619 interact with the last primer-template base-pair. Tyr619 interacts via a water-mediated hydrogen bond with the phosphate of the terminal primer nucleotide. The main-chain amide of Tyr391 interacts with the corresponding template nucleotide. A hydrogen bond has been postulated between Tyr391 and the hydroxyl group of Tyr567, a residue that plays a key role in base discrimination. This hydrogen bond may be crucial for forcing an infrequent Tyr567 rotamer conformation and, when the bond is removed, may influence fidelity. We investigated the roles of these residues in replication fidelity in vivo employing phage T4 rII reversion assays and an rI forward assay. Tyr391 was replaced by Phe, Met and Ala, and Tyr619 by Phe. The Y391A mutant, reported previously to decrease polymerase affinity for incoming nucleotides, was unable to support DNA replication in vivo, so we used an in vitro fidelity assay. Tyr391F/M replacements affect fidelity only slightly, implying that the bond with Tyr567 is not essential for fidelity. The Y391A enzyme has no mutator phenotype in vitro. The Y619F mutant displays a complex profile of impacts on fidelity but has almost the same mutational spectrum as the parental enzyme. The Y619F mutant displays reduced DNA binding, processivity, and exonuclease activity on single-stranded DNA and double-stranded DNA substrates. The Y619F substitution would disrupt the hydrogen bond network at the primer terminus and may affect the alignment of the 3' primer terminus at the polymerase active site, slowing chemistry and overall DNA synthesis. 相似文献
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The analysis of the active site region in the crystal structures of template-primer-bound KlenTaq (Klenow fragment equivalent of Thermus aquaticus polymerase I) shows the presence of an approximately 18-A long H-bonding track contributed by the Klenow fragment equivalent of Asn(845), Gln(849), Arg(668), His(881), and Gln(677). Its location is nearly diagonal to the helical axis of the template-primer. Four base pairs in the double stranded region proximal to 3' OH end of the primer terminus appear to interact with individual amino acid components of the track through either the bases or sugar moieties. To understand the functional significance of this H-bonding network in the catalytic function of Klenow fragment (KF), we generated N845A, N845Q, Q849A, Q849N, R668A, H881A, H881V, Q677A, and Q677N mutant species by site-directed mutagenesis. All of the mutant enzymes showed low catalytic activity. The kinetic analysis of mutant enzymes indicated that K(m)(.dNTP) was not significantly altered, but K(D)(.DNA) was significantly increased. Thus the mutant enzymes of the H-bonding track residues had decreased affinity for template-primer, although the extent of decrease was variable. Most interestingly, even the reduced binding of TP by the mutant enzymes occurs in the nonproductive mode. These results demonstrate that an H-bonding track is necessary for the binding of template-primer in the catalytically competent orientation in the pol I family of enzymes. The examination of the interactive environment of individual residues of this track further clarifies the mode of cooperation in various functional domains of pol I. 相似文献
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The mechanism of the 3'-5' exonuclease activity of the Klenow fragment of DNA polymerase I has been investigated with a combination of biochemical and spectroscopic techniques. Site-directed mutagenesis was used to make alanine substitutions of side chains that interact with the DNA substrate on the 5' side of the scissile phosphodiester bond. Kinetic parameters for 3'-5' exonuclease cleavage of single- and double-stranded DNA substrates were determined for each mutant protein in order to probe the role of the selected side chains in the exonuclease reaction. The results indicate that side chains that interact with the penultimate nucleotide (Q419, N420, and Y423) are important for anchoring the DNA substrate at the active site or ensuring proper geometry of the scissile phosphate. In contrast, side chains that interact with the third nucleotide from the DNA terminus (K422 and R455) do not participate directly in exonuclease cleavage of single-stranded DNA. Alanine substitutions of Q419, Y423, and R455 have markedly different effects on the cleavage of single- and double-stranded DNA, causing a much greater loss of activity in the case of a duplex substrate. Time-resolved fluorescence anisotropy decay measurements with a dansyl-labeled primer/template indicate that the Q419A, Y423A, and R455A mutations disrupted the ability of the Klenow fragment to melt duplex DNA and bind the frayed terminus at the exonuclease site. In contrast, the N420A mutation stabilized binding of a duplex terminus to the exonuclease site, suggesting that the N420 side chain facilitates the 3'-5' exonuclease reaction by introducing strain into the bound DNA substrate. Together, these results demonstrate that protein side chains that interact with the second or third nucleotides from the terminus can participate in both the chemical step of the exonuclease reaction, by anchoring the substrate in the active site or by ensuring proper geometry of the scissile phosphate, and in the prechemical steps of double-stranded DNA hydrolysis, by facilitating duplex melting. 相似文献
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To identify the sites in the Klenow fragment of Escherichia coli DNA polymerase I that interact with the ssDNA overhang of the template strand in the pre-polymerase ternary complex, we carried out UV-mediated photo-cross-linking of the enzyme-DNA-dNTP ternary complex. The template strand contained a nine-nucleotide overhang and was radiolabeled at the 5'-end. Since the enzyme-TP-dNTP ternary complex but not the E-TP binary complex is stable at high ionic strengths, the cross-linking was carried out in the presence of 0.5 M NaCl. The cross-linked E-TP-dNTP complex was purified and subjected to trypsin digestion. The radiolabeled TP cross-linked peptide was further purified by DEAE-Sepharose and C18 column chromatography and subjected to amino acid sequencing. The release of radiolabeled DNA during each sequencing cycle was also monitored. The sequencing results as well as the radioactivity release pattern show that F771, contained in a peptide spanning amino acids 759-775 of pol I, is the unequivocal site of the template cross-linking. A qualitative assessment of the cross-linking efficiency of the template overhang containing a TT sequence at different positions in the ternary complex further suggests that the major cross-linking site within the template overhang is at the second and/or third nucleotide. An examination of the F771A mutant enzyme showed that it was able to form the E-TP binary as well as E-TP-dNTP ternary complex; however, it could not cross-link to the template-primer in the ternary complex. Furthermore, the ternary complex with F771A was qualitatively defective and exhibited some salt sensitivity. These results suggest that F771 participates in the stabilization of the pre-polymerase ternary complex. 相似文献
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G. S. Shatskaya V. L. Drutsa O. N. Koroleva I. A. Osterman T. M. Dmitrieva 《Biochemistry. Biokhimii?a》2013,78(1):96-101
The activities of wild-type mengovirus RNA polymerase (RdRP) and of its three mutants with C-terminal tryp-tophan residue replaced by residues of alanine (W460A), phenylalanine (W460F), or tyrosine (W460Y) were studied. The proteins were expressed in E. coli and purified by affinity chromatography with the IMPACT system. The isolated recombinant proteins were studied using a cell-free replication system on elongation of oligo(U) primer on RNA template corresponding to the 3′-terminal 366-meric fragment of the mengovirus RNA. The activities of the mutant polymerases were comparable to that of the wild-type enzyme. 相似文献
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Najmudin S Coté ML Sun D Yohannan S Montano SP Gu J Georgiadis MM 《Journal of molecular biology》2000,296(2):613-632
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Phi29 DNA polymerase residues Tyr59, His61 and Phe69 of the highly conserved ExoII motif are essential for interaction with the terminal protein
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Phage Φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3′→5′ exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, Φ29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3′→5′ exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of Φ29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction. 相似文献
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Recognition and binding of template-primers containing defined abasic sites by Drosophila DNA polymerase alpha holoenzyme 总被引:2,自引:0,他引:2
Human DNA polymerase alpha holoenzyme follows an ordered sequential terreactant mechanism of substrate recognition and binding (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S.-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We confirmed this mechanism for the DNA polymerase alpha holoenzyme purified from Drosophila melanogaster embryos and studied the interaction of Drosophila pol alpha with synthetic oligonucleotide template-primers containing modified tetrahydrofuran moieties as model abasic lesions chemically engineered at a number of defined sites. Abasic lesions in the template had relatively little effect on the polymerase incorporation reaction at sites proximal to the lesion. However, incorporation opposite an abasic site was undetectable relative to that which occurred opposite a normal template nucleotide. Moreover, abasic residues in the primer region of the template-primer construct as far as 4 base pairs removed from the 3'-primer terminus prevented detectable nucleotide incorporation relative to that seen on an unmodified template-primer. Primer-region lesions had qualitatively similar effects whether they were located on the primer strand itself or on the complementary template strand. Data from polymerase incorporation experiments were corroborated by competitive binding assays performed under steady state reaction conditions. Results of these experiments suggested that polymerase binding to synthetic oligonucleotide template-primers was essentially unaffected by lesions located at sites that did not block incorporation. Lesions that did block incorporation apparently did so by abrogating template-primer binding. These observations have implications for understanding the mechanisms whereby DNA polymerase alpha recognizes noninformational template sites in vivo and prevents DNA synthesis from proceeding past these points. 相似文献
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Joseph M. Dahl Hongyun Wang José M. Lázaro Margarita Salas Kate R. Lieberman 《The Journal of biological chemistry》2014,289(10):6350-6361
The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn2+ rather than Mg2+. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture. 相似文献
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The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis. 相似文献