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C M Joyce

Publications and source records attributed to C M Joyce.

At least 19 recordsLinked to original sources
PubMed1998

How E. coli DNA polymerase I (Klenow fragment) distinguishes between deoxy- and dideoxynucleotides.

Deoxy- and dideoxynucleotides differ only in whether they have a hydroxyl substituent at C-3' of the ribose moiety, and yet the Klenow fragment DNA polymerase prefers the natural (dNTP) substrate by several thousandfold. We have used this preference in order to investigate how Klenow fragment interacts with the sugar portion of an incoming dNTP. We screened mutant derivatives of Klenow fragment so as to identify those amino acid residues that play important roles in distinguishing between dNTPs and ddNTPs. Substitution of Phe762 with Ala or Tyr caused a dramatic decrease in the discrimination against ddNTPs, while mutations in Tyr766 and Glu710 had a smaller effect, suggesting that these two side-chains play secondary roles in the selection of dNTPs over ddNTPs. In order to understand the interactions in the enzyme-DNA-dNTP ternary complex, pre-steady-state kinetic parameters for the incorporation of dNTPs and ddNTPs were determined for wild-type Klenow fragment and for mutant derivatives that showed changes in dNTP/ddNTP discrimination. From elemental effect measurements we infer that selection against dideoxynucleotides takes place in the transition state for the conformational change that precedes phosphoryl transfer. The crucial role of the Phe762 side-chain appears to be to constrain the dNTP molecule so that the 3'-OH can make an interaction with another group within the ternary complex. When Tyr is substituted at position 762, the same interactions can take place to position the dNTP, but specificity against the ddNTP is lost because the phenolic OH can compensate for the missing 3'-OH of the nucleotide. Substitution of the smaller Ala side-chain results in a loss in specificity because the dNTP is no longer appropriately constrained. Measurement of reaction rates as a function of magnesium ion concentration suggests that the interaction made with the dNTP 3'-OH may involve a metal ion and the Glu710 side-chain, the simplest scenario being that both the 3'-OH and the carboxylate of Glu710 are ligands to the same metal ion.

Amino Acid Sequence↗
PubMed1998

A single side chain prevents Escherichia coli DNA polymerase I (Klenow fragment) from incorporating ribonucleotides.

Although nucleic acid polymerases from different families show striking similarities in structure, they maintain stringent specificity for the sugar structure of the incoming nucleoside triphosphate. The Klenow fragment of E. coli DNA polymerase I selects its natural substrates, deoxynucleotides, over ribonucleotides by several thousand fold. Analysis of mutant Klenow fragment derivatives indicates that discrimination is provided by the Glu-710 side chain which sterically blocks the 2'-OH of an incoming rNTP. A nearby aromatic side chain, at position 762, plays an important role in constraining the nucleotide so that the Glu-710 "steric gate" can be fully effective. Even with the E710A mutation, which is extremely permissive for addition of a single ribonucleotide to a DNA primer, Klenow fragment does not efficiently synthesize pure RNA, indicating that additional barriers prevent the incorporation of successive ribonucleotides.

Binding Sites↗
PubMed1998

Use of fluorescence resonance energy transfer to investigate the conformation of DNA substrates bound to the Klenow fragment.

Fluorescence resonance energy transfer (FRET) has been used to investigate the conformation of the single stranded region for a series of fluorescent DNA template-primers bound to the Klenow fragment (KF) of Escherichia coli DNA polymerase I. Fluorescent derivatives of template-primer DNA, modified with tetramethylrhodamine (TMR), served as energy transfer acceptors to the donor fluorescein fluorophore used to modify cysteine 751 in the double mutant KF (S751C, C907S). Design of the template-primer allowed the probe's position within the DNA-protein complex to be varied by stepwise extension of the primer strand upon addition of the appropriate deoxynucleoside triphosphates (dNTP). The TMR acceptor probe occupied seven different positions in the template-primers, five in the single stranded region and two in the double stranded region. The efficiency of energy transfer was determined at each position by calculating the integrated area of the fluorescein emission peak in the presence and absence of acceptor. Results indicate that the FRET efficiency varied in a sinusoidal fashion with a periodicity of approximately 10 base pairs and that the data could be fitted to an equation derived from a simple model formulated on the basis of helical structure. The data support the conclusion that the single stranded template portion of a DNA template-primer adopts a helical conformation when bound to the KF. The results of this study further support FRET as a useful method for the determination of structure and conformation in protein-DNA complexes.

DNA Polymerase I↗
PubMed1998

Effects of mutations on the partitioning of DNA substrates between the polymerase and 3'-5' exonuclease sites of DNA polymerase I (Klenow fragment).

Site-directed mutagenesis and time-resolved fluorescence spectroscopy were used to evaluate the contributions of individual amino acid side chains to the binding of DNA primer-templates to the 3'-5' exonuclease site of the large proteolytic fragment (Klenow fragment) of DNA polymerase I. Mutations were introduced into side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease site. The wild-type residues were replaced by alanine in each case. To assess the effects of the mutations on DNA binding, time-resolved fluorescence anisotropy measurements were performed on dansyl-labeled primer-templates bound to the mutant enzymes. In contrast to techniques that simply monitor the overall binding of proteins to DNA, the time-resolved fluorescence anisotropy technique was used to determine the fractional occupancies of the polymerase and 3'-5' exonuclease active sites of Klenow fragment. Equilibrium constants describing the partitioning of DNA between the two active sites were obtained for nine different mutant enzymes bound to both matched and mismatched DNA sequences. Mutations of Leu361 and Phe473 caused the largest effects, significantly destabilizing the binding of mismatched DNA substrates to the 3'-5' exonuclease site relative to DNA bound at the polymerase site, consistent with structural data showing that the side chains of these residues are involved in intimate hydrophobic interactions with the 3' terminal and penultimate bases of the primer strand [Beese, L., and Steitz, T. A. (1991) EMBO J. 10, 25-33]. Mutations of the His660 and Glu357 side chains also resulted in significant effects on the binding of mismatched DNA to the 3'-5' exonuclease site. Surprisingly, mutation of Tyr497 increased the partitioning of mismatched DNA into the 3'-5' exonuclease site, suggesting that the tyrosine side chain in the wildtype enzyme destabilizes substrate binding, despite crystallographic data showing that Tyr497 is H-bonded to the DNA substrate. The effects of mutating the amino acid side chains that serve as ligands to two divalent metal ions bound at the 3'-5' exonuclease site, designated A and B, indicated that metal A also helps to bind DNA to the 3'-5' exonuclease site. These results demonstrate that the time-resolved fluorescence anisotropy technique can be used to quantify the energetic contributions associated with each of the crystallographically defined DNA-protein contacts at the 3'-5' exonuclease site.

Amino Acid Substitution↗
PubMed1997

Biochemical and mutational studies of the 5'-3' exonuclease of DNA polymerase I of Escherichia coli.

In order to improve our understanding of the 5'-3' exonuclease reaction catalyzed by Escherichia coli DNA polymerase I, we have constructed expression plasmids and developed purification methods for whole DNA polymerase I and its 5'-3' exonuclease domain that allow the production of large quantities of highly purified material suitable for biophysical and other studies. We have studied the enzymatic properties of the 5'-3' exonuclease, both as an isolated domain and in the context of the whole polymerase, using a variety of model oligonucleotides to explore the enzyme-substrate interaction. The 5'-3' exonuclease is known to be a structure-specific nuclease that cleaves a 5' displaced strand at the junction between single-stranded and duplex regions. Since the isolated domain shows the same structure specificity as the whole polymerase, the correct geometry of substrate binding is achieved without the assistance of the polymerase domain. The 5'-3' exonuclease reaction has a strict requirement for a free 5' end on the displaced strand; however, the upstream template and primer strands are dispensable. Site-directed mutagenesis of the ten carboxylate residues that are highly conserved among bacterial and bacteriophage 5'-3' exonucleases indicates that nine of them are important in the reaction. This finding is discussed in relation to structural and mutational data for related 5' nucleases.

Amino Acid Sequence↗
PubMed1997

Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O helix of the fingers subdomain.

A mutant derivative of Klenow fragment DNA polymerase containing serine substituted for tyrosine at residue 766 has been shown by kinetic analysis to have an increased misinsertion rate relative to wild-type Klenow fragment, but a decreased rate of extension from the resulting mispairs (Carroll, S. S., Cowart, M., and Benkovic, S. J. (1991) Biochemistry 30, 804-813). In the present study we use an M13mp2-based fidelity assay to study the error specificity of this mutator polymerase. Despite its compromised ability to extend mispairs, the Y766S polymerase and a Y766A mutant both have elevated base substitution error rates. The magnitude of the mutator effect is mispair-specific, from no effect for some mispairs to rates elevated by 60-fold for misincorporation of TMP opposite template G. The results with the Y766S mutant are remarkably consistent with the earlier kinetic analysis of misinsertion, demonstrating that either approach can be used to identify and characterize mutator polymerases. Both the Y766S and Y766A mutant polymerases are also frameshift mutators, having elevated rates for two-base deletions and a 276-base deletion between a direct repeat sequence. However, neither mutant polymerase has an increased error rate for single-base frameshifts in repetitive sequences. This error specificity suggests that the deletions generated by the mutator polymerases are initiated by misinsertion rather than by strand slippage. When considered with recent structure-function studies of other polymerases, the data indicate that the nucleotide misinsertion and strand-slippage mechanisms for polymerization infidelity are differentially affected by changes in distinct structural elements of DNA polymerases that share similar subdomain structures.

Base Sequence↗
PubMed1996

A thumb subdomain mutant of the large fragment of Escherichia coli DNA polymerase I with reduced DNA binding affinity, processivity, and frameshift fidelity.

In Klenow fragment DNA polymerase, a flexible 50-amino acid subdomain at the tip of the thumb which includes two alpha helices has been suggested to interact with the duplex template-primer (Beese, L.S., Derbyshire, V. and Steitz, T.A. (1993) Science 260, 352-355). The present study investigates the properties of Klenow polymerase containing a 24-amino acid deletion (residues 590-613) that removes a portion of the tip of the thumb. The mutant polymerase has relatively normal dNTP binding and catalytic rate. However, its DNA binding affinity is reduced by more than 100-fold relative to the intact polymerase and its ability to conduct processive synthesis is also reduced. Although the mutant polymerase has relatively normal base substitution fidelity, it has strongly reduced frameshift fidelity, being especially error-prone for single nucleotide addition errors in homopolymeric runs. The addition error rate increases as the length of the reiterated sequence increases, indicative of errors initiated by template-primer strand slippage. These observations suggest a role for the tip of the thumb of Klenow polymerase in determining DNA binding, processivity and frameshift fidelity, perhaps by tracking the minor groove of the duplex DNA. The results are discussed in light of remarkably similar observations with T7 DNA polymerase in the presence or absence of thioredoxin, an accessory subunit that affects these same properties.

Base Sequence↗
PubMed1995

Deoxynucleoside triphosphate and pyrophosphate binding sites in the catalytically competent ternary complex for the polymerase reaction catalyzed by DNA polymerase I (Klenow fragment).

We have employed site-directed mutagenesis to identify those amino acid residues that interact with the deoxynucleoside triphosphate (dNTP) and pyrophosphate in the Klenow fragment-DNA-dNTP ternary complex. Earlier structural, mutagenesis, and labeling studies have suggested that the incoming dNTP molecule contacts a region on one side of the polymerase cleft, primarily involving residues within the so-called "fingers" subdomain. We have made mutations in residues seen to be close to the dNTP in the crystal structure of the Klenow fragment-dNTP binary complex and have examined their kinetic parameters, particularly Km(dNTP). The results are consistent with the notion that there are significant differences between the dNTP interactions in the binary and ternary complexes, although some contacts may be present in both. When dTTP is the incoming nucleotide, the side chains of Arg754 and Phe762 make the largest contributions to binding; measurement of Km(PPi) suggests that Arg754 contacts the beta- or gamma-phosphate of the dNTP. With dGTP, the contribution of Arg754 remains the same, but the additional interactions are provided by both Lys758 and Phe762, suggesting that the binding of the incoming dNTP is not identical under all circumstances. Mutations in Arg754 and Lys758 also cause a substantial decrease in the rate of polymerase-catalyzed incorporation, and sulfur elemental effect measurements indicate that loss of Arg754 (and perhaps also Lys758) slows the rate of the chemical step of the reaction. Mutations of Arg682, His734, and Tyr766 affect the binding of DNA, suggesting that these mutations, whose effect on dNTP binding is small, may influence dNTP binding indirectly via the positioning of the DNA template-primer.

Amino Acid Sequence↗
PubMed1994

Characterization of four new beta-tubulin genes and their expression during male flower development in maize (Zea mays L.).

Four different beta-tubulin coding sequences were isolated from a cDNA library prepared from RNA from maize seedling shoots. The four genes (designated tub4, tub6, tub7 and tub8) represented by these cDNA clones together with the tub1 and tub2 genes reported previously encode six beta-tubulin isotypes with 90-97.5% amino acid sequence identity. Results from phylogenetic analysis of 17 beta-tubulin genes from monocot and dicot plant species indicated that multiple extant lines of beta-tubulin genes diverged from a single precursor after the appearance of the two major subfamilies of alpha-tubulin genes described previously. Hybridization probes from the 3' non-coding regions of six beta-tubulin clones were used to quantify the levels of corresponding tubulin transcripts in different maize tissues including developing anthers and pollen. The results from these dot blot hybridization experiments showed that all of the beta-tubulin genes were expressed in most tissues examined, although each gene showed a unique pattern of differential transcript accumulation. The tub1 gene showed a high level of transcript accumulation in meristematic tissues and almost no accumulation in the late stages of anther development and in pollen. In contrast, the level of tub4 transcripts was very low during early stages of male flower development but increased markedly (more than 100 times) during the development of anthers and in pollen. Results from RNAse protection assays showed that this increased hybridization signal resulted from expression of transcripts from one or two genes closely related to tub4. The tub4-related transcripts were not present in shoot tissue. Transcripts from the tub2 gene accumulated to very low levels in all tissues examined, but reached the highest levels in young anthers containing microspore mother cells. RNAse protection assays were used to measure the absolute levels of alpha- and beta-tubulin transcripts in seedling shoot and in pollen. The alpha-tubulin gene subfamily I genes (tua1, tua2, tua4) contributed the great majority of alpha-tubulin transcripts in both shoot and pollen. Transcripts from the beta-tubulin genes tub4, tub6, tub7, and tub8 were predominant in shoot, but were much less significant than the tub4-related transcripts in pollen.

Amino Acid Sequence↗
PubMed1993

Re-engineering the polymerase domain of Klenow fragment and evaluation of overproduction and purification strategies.

We describe experiments to produce large quantities of the polymerase domain of E. coli DNA polymerase I for biochemical and biophysical studies. The polymerase domain derivative used in previous studies was insoluble when overproduced and tended to aggregate during purification. These problems were solved by a combination of two distinct strategies. By changing the expression system, we were able to obtain the overproduced protein in a soluble form, a necessary first step since attempts to purify the polymerase domain from the insoluble pellet were unsuccessful. The tendency of the polymerase domain to aggregate was eliminated by re-engineering the protein so as to remove both a solvent-exposed hydrophobic patch and a potentially unstructured region at the extreme N-terminus. Unlike the original construct, the re-engineered derivatives chromatographed as a single species and could be purified to homogeneity in good yield. Our experience in this study emphasizes the level of ignorance of the factors that influence protein overproduction and the need, in difficult cases, to evaluate many strategies in a semi-empirical manner.

Amino Acid Sequence↗
PubMed1992

Reactions at the polymerase active site that contribute to the fidelity of Escherichia coli DNA polymerase I (Klenow fragment).

In order to study the structural principles governing DNA polymerase fidelity we have measured the rates of insertion of incorrect nucleotides and the rates of extension from the resulting mismatched base pairs, catalyzed by the Klenow fragment of DNA polymerase I. Using a combination of semi-quantitative and qualitative approaches, we have studied each of the 12 possible mismatches in a variety of sequence contexts. The results indicate that Klenow fragment discriminates between mismatches largely on the basis of the identity of the mismatch, with the surrounding sequence context playing a significant, but secondary, role. For purine-pyrimidine and pyrimidine-pyrimidine mispairs, the relative ease of mismatch synthesis and extension can be rationalized using a simple geometrical model, with the important criterion being the extent to which the mismatched base pair can conform to normal DNA geometry. Essentially similar conclusions have been reached in studies of other polymerases, suggesting that this aspect of mispair geometry is sensed and responded to in a similar way by all polymerases. Purine-purine mismatches form a less cohesive class, showing more variable behavior from mispair to mispair, and a greater apparent susceptibility to sequence context effects. Comparison of our data with studies of other polymerases also suggests that different polymerases respond to purine-purine mismatches in distinct and characteristic ways. An extensive analysis of each of the four purine-purine mispairs in approximately 100 different sequence contexts suggests that the reaction is influenced both by the local DNA structure and by the ability of the mismatched terminus to undergo slippage.

Base Composition↗
PubMed1992

Alpha-tubulin gene family of maize (Zea mays L.). Evidence for two ancient alpha-tubulin genes in plants.

Among 81 alpha-tubulin cDNA clones prepared from RNA from maize seedling shoot, endosperm and pollen, we identified six different alpha-tubulin coding sequences. The DNA sequence analysis of coding and non-coding regions from the clones showed that they can be classified into three different alpha-tubulin gene subfamilies. Genes within each subfamily encode proteins that are 99 to 100% identical in amino acid sequence. Deduced amino acid sequence identity between genes in different subfamilies ranges from 89 to 93%. The results of hybridizations of genomic DNAs to alpha-tubulin coding region probes and to 3' non-coding region probes constructed from six different alpha-tubulin cDNA clones indicated that the maize alpha-tubulin gene family contains at least eight members. Comparison of deduced alpha-tubulin amino acid sequences from maize and the dicot species Arabidopsis thaliana showed that alpha-tubulin isotypes encoded by genes in maize subfamilies I and II are more similar to specific Arabidopsis gene products (96 to 97% amino acid identity) than to isotypes encoded by genes in the other maize subfamilies. Phylogenetic analyses revealed that genes in these two subfamilies were derived from two ancient alpha-tubulin genes that predate the divergence of monocots and dicots. These same analyses revealed that the gene in maize subfamily III is more closely related to sequences from subfamily I genes than to those from subfamily II genes. However, the subfamily III gene has no close counterpart in Arabidopsis. We found evidence of a transposable element-like insertion in the subfamily III gene in some maize lines.

Base Sequence↗