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1.
Several variants of RB69 DNA polymerase (RB69 pol) with single-site replacements in the nascent base-pair binding pocket are less discriminating with respect to noncomplementary dNMP incorporation than the wild-type enzyme. To quantify the loss in base selectivity, we determined the transient-state kinetic parameters for incorporation of correct and all combinations of incorrect dNMPs by the exonuclease-deficient form of one of these RB69 pol variants, L561A, using rapid chemical quench assays. The L561A variant did not significantly alter the k(pol) and K(D) values for incorporation of correct dNMPs, but it showed increased incorporation efficiency (k(pol)/K(D)) for mispaired bases relative to the wild-type enzyme. The incorporation efficiency for mispaired bases by the L561A variant ranged from 1.5 x 10(-)(5) microM(-)(1) s(-)(1) for dCMP opposite templating C to 2 x 10(-)(3) microM(-)(1) s(-)(1) for dAMP opposite templating C. These k(pol)/K(D) values are 3-60-fold greater than those observed with the wild-type enzyme. The effect of the L561A replacement on the mutation frequency in vivo was determined by infecting Escherichia coli harboring a plasmid encoding the L561A variant of RB69 pol with T4 phage bearing a mutant rII locus, and the rates of reversions to rII(+) were scored. The exonuclease-proficient RB69 pol L561A displayed a weak mutator phenotype. In contrast, no progeny phage were produced after infection of E. coli, expressing an exonuclease-deficient RB69 pol L561A, with either mutant or wild-type T4 phage. This dominant-lethal phenotype was attributed to error catastrophe caused by the high rate of mutation expected from combining the pol L561A and exo(-) mutator activities. 相似文献
2.
Xia S Wang M Lee HR Sinha A Blaha G Christian T Wang J Konigsberg W 《Journal of molecular biology》2011,406(4):558-10149
We have previously observed that stepwise replacement of amino acid residues in the nascent base-pair binding pocket of RB69 DNA polymerase (RB69pol) with Ala or Gly expanded the space in this pocket, resulting in a progressive increase in misincorporation. However, in vivo results with similar RB69pol nascent base-pair binding pocket mutants showed that mutation rates, as determined by the T4 phage rI forward assay and rII reversion assay, were significantly lower for the RB69pol S565G/Y567A double mutant than for the Y567A single mutant, the opposite of what we would have predicted. To investigate the reasons for this unexpected result, we have determined the pre-steady-state kinetic parameters and crystal structures of relevant ternary complexes. We found that the S565G/Y567A mutant generally had greater base selectivity than the Y567A mutant and that the kinetic parameters for dNMP insertion, excision of the 3′-terminal nucleotide residue, and primer extension beyond a mispair differed not only between these two mutants but also between the two highly mutable sequences in the T4 rI complementary strand. Comparison of the crystal structures of these two mutants with correct and incorrect incoming dNTPs provides insight into the unexpected increase in the fidelity of the S565G/Y567A double mutant. Taken together, the kinetic and structural results provide a basis for integrating and interpreting in vivo and in vitro observations. 相似文献
3.
噬菌体RB69外切酶活性缺失的DNA 聚合酶突变体(D222A/D327A)在大肠杆菌细胞中表达,表达量达细胞蛋白总量的69% .表达后经DEAE-Sepharose FastFlow , Source 30Q 和HTP三步分离纯化,纯度可达99% 以上.随后测定了该酶利用5种dNTP为底物进行聚合反应的酶促动力学常数(Km 和Kcat),结果表明该酶利用dUTP的能力与利用dTTP的能力相近,Km (dTTP)和Km(dUTP)均较高于其它3种脱氧核苷酸的Km (dATP, dCTP, dGTP),推测其Km 值的差异主要来源于T/U 碱基本身,而并非全部由GC碱基配对与AT碱基配对之间的氢键作用力的强弱差别所决定. 相似文献
4.
Many DNA polymerases select their natural substrates, deoxy- as opposed to ribonucleoside triphosphates, with a selectivity greater than 10000-fold. The function of a highly conserved residue, Tyr416, in the palm domain of the parental enzyme, an exo(-) derivative of RB69 DNA polymerase (gp43), a member of the pol alpha DNA polymerase family, was examined for its role in helping the polymerase discriminate between ribo-, dideoxyribo-, and deoxyribonucleoside triphosphates. The parental enzyme selected dNTPs vs rNTPs with about the same preference as dNTPs vs ddNTPs. Pre-steady-state kinetic analysis was carried out with the parental enzyme and two mutants, Y416A and Y416F. The Y416A mutant incorporated ribonucleotide residues much more efficiently than the parental enzyme, whereas the Y416F mutant was more permissive toward ddNTP vs rNTP utilization than either the Y416A mutant or the parental enzyme. We also found that both dCDP and rCDP inhibited dCTP incorporation by the Y416A mutant, while only dCDP but not rCDP inhibited dCTP incorporation by the parental enzyme and the Y416F mutant. The parental enzyme and the Y416A and Y416F mutants were all able to add araCTP (1-beta-D-arabinofuranosylcytosine-5'-triphosphate) to a primer but with reduced efficiency relative to dCTP. Based on our kinetic results, interpreted in the context of the crystal structure of the RB69 gp43 ternary complex, we suggest that sugar discrimination is provided mainly by the Tyr416 side chain which can sterically block the 2'-OH group of an incoming rNTP. 相似文献
5.
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
6.
Residues in the nascent base pair binding pocket (NBP) of bacteriophage RB69 DNA polymerase (RB69pol) are responsible for base discrimination. Replacing Tyr567 with Ala leads to greater flexibility in the NBP, increasing the probability of misincorporation. We used the fluorescent cytosine analogue, 1,3-diaza-2-oxophenoxazine (tC(o)), to identify preinsertion step(s) altered by NBP flexibility. When tC(o) is the templating base in a wild-type (wt) RB69pol ternary complex, its fluorescence is quenched only in the presence of dGTP. However, with the RB69pol Y567A mutant, the fluorescence of tC(o) is also quenched in the presence of dATP. We determined the crystal structure of the dATP/tC(o)-containing ternary complex of the RB69pol Y567A mutant at 1.9 ? resolution and found that the incoming dATP formed two hydrogen bonds with an imino-tautomerized form of tC(o). Stabilization of the dATP/tC(o) base pair involved movement of the tC(o) backbone sugar into the DNA minor groove and required tilting of the tC(o) tricyclic ring to prevent a steric clash with L561. This structure, together with the pre-steady-state kinetic parameters and dNTP binding affinity, estimated from equilibrium fluorescence titrations, suggested that the flexibility of the NBP, provided by the Y567 to Ala substitution, led to a more favorable forward isomerization step resulting in an increase in dNTP binding affinity. 相似文献
7.
Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol. 相似文献
8.
Trzemecka A Jacewicz A Carver GT Drake JW Bebenek A 《Journal of molecular biology》2010,404(5):778-793
Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article. 相似文献
9.
The kinetics of forming a proper Watson–Crick base pair as well incorporating bases opposite furan, an abasic site analog, have been well characterized for the B Family replicative DNA polymerase from bacteriophage T4. Structural studies of these reactions, however, have only been performed with the homologous enzyme from bacteriophage RB69. In this work, the homologous enzymes from RB69 and T4 were compared in parallel reactions to determine the relative abilities of the two polymerases to incorporate correct nucleotides as well as to form improper pairings. The kinetic rates for three different exonuclease mutants for each enzyme were measured for incorporation of an A opposite T and an A opposite furan as well as for the formation of A:C and T:T mismatches. The T4 exonuclease mutants were all ~2- to 7-fold more efficient than the corresponding RB69 exonuclease mutants depending on whether a T or furan was in the templating position and which exonuclease mutant was used. The rates for mismatch formation by T4 were significantly reduced compared with incorporation opposite furan, much more so than the corresponding RB69 mutant. These results show that there are kinetic differences between the two enzymes but they are not large enough to preclude structural assumptions for T4 DNA polymerase based on the known structure of the RB69 DNA polymerase. 相似文献
10.
Site specific mutants in the pol active center of RB69 DNA polymerase have been produced and studied using rapid chemical-quench techniques. Pre-steady-state kinetic analysis carried out with Mg(2+) and Mn(2+) has enabled us to divide the mutants into two groups. One group had greatly reduced k(pols) values in the presence of Mg(2+) but responded to Mn(2+) which restored the k(pol) values for the nucleotidyl transfer reaction to near wild-type levels. The other group of mutants also had lower k(pol) values, relative to that of the wild-type polymerase, but could not be rescued by Mn(2+). The behavior of these mutants was interpreted in terms of the crystal structures of the available RB69 pol complexes. Our results on the metal ion dependence of the D621A and E686A mutants, together with knowledge of the position of their side chains in two different RB69 pol conformations, suggest that these acidic residues serve as alternative ligands for the metal ions destined to occupy the A and B catalytic sites. We infer that this occurs prior to the conformational change that produces the ternary RB69 pol complex in which the A and B metal ions are ligated by D623 and D411 as the enzyme is poised for phosphoryl transfer. 相似文献
11.
Shuangluo Xia Marcus Wood Michael J. Bradley Enrique M. De La Cruz William H. Konigsberg 《Nucleic acids research》2013,41(19):9077-9089
Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tCo-tCnitro Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography. 相似文献
12.
Engineering Streptomyces clavuligerus deacetoxycephalosporin C synthase for optimal ring expansion activity toward penicillin G 总被引:1,自引:0,他引:1
Wei CL Yang YB Wang WC Liu WC Hsu JS Tsai YC 《Applied and environmental microbiology》2003,69(4):2306-2312
The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k(cat)/K(m) ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k(cat)/K(m) values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k(cat)/K(m) ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction. 相似文献
13.
We have recently challenged the widely held view that 2,4-difluorotoluene (dF) is a nonpolar isosteric analogue of the nucleotide dT, incapable of forming hydrogen bonds (HBs). To gain a further understanding for the kinetic preference that favors dAMP insertion opposite a templating dF, a result that mirrors the base selectivity that favors dAMP insertion opposite dT by RB69 DNA polymerase (RB69pol), we determined presteady-state kinetic parameters for incorporation of four dNMPs opposite dF by RB69pol and solved the structures of corresponding ternary complexes. We observed that both the F2 and F4 substituent of dF in these structures serve as HB acceptors forming HBs either directly with dTTP and dGTP or indirectly with dATP and dCTP via ordered water molecules. We have defined the shape and chemical features of each dF/dNTP pair in the RB69pol active site without the corresponding phosphodiester-linkage constraints of dF/dNs when they are embedded in isolated DNA duplexes. These features can explain the kinetic preferences exhibited by the templating dF when the nucleotide incorporation is catalyzed by wild type RB69pol or its mutants. We further show that the shapes of the dNTP/dF nascent base pair differ markedly from the corresponding dNTP/dT in the pol active site and that these differences have a profound effect on their incorporation efficiencies. 相似文献
14.
Steady-state kinetic characterization of RB69 DNA polymerase mutants that affect dNTP incorporation.
The function of six highly conserved residues (Arg482, Lys483, Lys486, Lys560, Asn564, and Tyr567) in the fingers domain of bacteriophage RB69 DNA polymerase (RB69 gp43) were analyzed by kinetic studies with mutants in which each of these residues was replaced with Ala. Our results suggest that Arg482, Lys486, Lys560, and Asn564 contact the incoming dNTP during the nucleotidyl transfer reaction as judged by variations in apparent Km and kcat values for dNTP incorporation by these mutants compared to those for the exonuclease deficient parental polymerase under steady-state conditions. On the basis of our studies, as well as on the basis of the crystal structure of RB69 gp43, we propose that a conformational change in the fingers domain, which presumably occurs prior to polymerization, brings the side chains of Arg482, Lys486, Lys560, and Asn564 into the vicinity of the primer-template terminus where they can contact the triphosphate moiety of the incoming dNTP. In particular, on the basis of structural studies reported for the "closed" forms of two other DNA polymerases and from the kinetic studies reported here, we suggest that (i) Lys560 and Asn564 contact the nonbonding oxygens of the alpha and beta phosphates, respectively, and (ii) both Arg482 and Lys486 contact the gamma phosphate oxygens of the incoming dNTP of RB69 gp43 prior to the nucleotidyl transfer reaction. We also found that Ala substitutions at each of these four RB69 gp43 sites could incorporate dGDP as a substrate, although with markedly reduced efficiency compared to that with dGTP. In contrast in the parental exo- background, the K483A and Y567A substituted enzymes could not use dGDP as a substrate for primer extension. These results, taken together, are consistent with the putative roles of the four conserved residues in RB69 gp43 as stated above. 相似文献
15.
16.
The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson's disease. In single-turnover kinetic studies, we show that the Y955C mutation results in a decrease in the maximal rate of polymerization and an increase in the K(m) for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM(-1) s(-1), respectively, representing reductions of 30-, 110-, and 1300-fold, respectively, relative to the value for the wild-type enzyme. The fidelity of incorporation was reduced 6-130-fold, largely because of the significant decrease in the specificity constant for correct dATP:T incorporation. For example, k(cat)/K(m) for forming a TTP:T mismatch was decreased 10-fold from 0.0002 to 0.00002 μM(-1) s(-1) by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to that of the wild type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, the level of incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role for Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of incorporation of dATP opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency. 相似文献
17.
Bebenek A Dressman HK Carver GT Ng S Petrov V Yang G Konigsberg WH Karam JD Drake JW 《The Journal of biological chemistry》2001,276(13):10387-10397
The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43. 相似文献
18.
19.
Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Br?nsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Br?nsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism. 相似文献
20.
In an attempt to generate mutant aminoacyl-tRNA synthetases capable of charging non-canonical amino acids, a series of yeast tyrosyl-tRNA synthetase (TyrRS) mutants was constructed by site-specific mutagenesis of putative active site residues, which were deduced by analogy with those of Bacillus stearothermophilus TyrRS. Among these mutants, one with the replacement of tyrosine at position 43 by glycine, "Y43G," was found to be able to utilize several 3-substituted tyrosine analogues as substrates for aminoacylation. The catalytic efficiency (k(cat)/K(m)) of mutant Y43G for aminoacylation with L-tyrosine was about 400-fold decreased as compared to that of the wild-type TyrRS. On the other hand, the ability to utilize 3-iodo-L-tyrosine was newly generated in this mutant TyrRS, since the wild-type TyrRS could not accept 3-iodo-L-tyrosine at all under physiological conditions. This mutant TyrRS should serve as a new tool for site-specific incorporation of non-canonical amino acids, such as those in 3-substituted tyrosine analogues, into proteins in an appropriate translation system in vivo or in vitro. 相似文献