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C M Joyce

Publications and source records attributed to C M Joyce.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Tubulin gene expression in maize (Zea mays L.). Change in isotype expression along the developmental axis of seedling root.

Two-dimensional gel/western blot analysis was used to characterize alpha- and beta-tubulin isotype expression along the developmental axis of the maize (Zea mays) seedling primary root. We identified four distinct alpha-tubulin isotypes and a minimum of six beta-tubulin isotypes. This analysis showed differences between the alpha- and beta-tubulin isotypes expressed in rapidly dividing tissue at the root tip and differentiated root tissues proximal to the tip. The alpha 1 and alpha 4 isotypes predominated in samples from immature rapidly dividing tissues such as root tips, whereas in mature tissues such as differentiated root and pollen, alpha 2, alpha 3 and alpha 4 isotypes predominated. The beta 1 and beta 2 isotypes were more abundant in protein samples from root cortex than in samples from the root tip or vascular cylinder. In contrast, the beta 4 and beta 5 isotypes appeared to be more abundant in root tip and vascular cylinder samples than in root cortex samples. Hybridization probes from the 3' non-coding region of six alpha-tubulin cDNA clones were used to quantify the levels of corresponding tubulin transcripts in selected tissues, from embryonic to mature and from largely undifferentiated to highly differentiated. The results from these hybridization experiments showed that all of the alpha-tubulin genes were expressed in all tissues examined, although each gene showed a unique pattern of differential transcript accumulation. A transcript produced from cDNA clone representing the tua5 alpha-tubulin gene was translated in vitro and produced an alpha-tubulin that comigrated with the alpha 2 isotype.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1992

Side chains involved in catalysis of the polymerase reaction of DNA polymerase I from Escherichia coli.

To continue our mutagenesis study of the polymerase active site of the Klenow fragment of DNA polymerase I, we have characterized new mutants with substitutions at Asp705, Glu710, and Glu883, and have investigated further the mutations in residues Arg668, Gln849, and Asp882, which showed strong Kcat effects in our previous study. To determine which step of the reaction is rate-limiting for each mutant protein, we measured the effect on the reaction rate of an alpha-thio-substituted dNTP. One group of mutants showed a substantial sulfur elemental effect, while a second group, like wild-type Klenow fragment, was unaffected by the phosphorothioate substitution. Consistent with earlier data, these results imply that, between formation of the enzyme-DNA-dNTP ternary complex and completion of phosphodiester bond formation, there are two kinetically distinct steps, only one of which is sensitive to sulfur substitution. The rather complex elemental effect data obtained with the mutant derivatives of Klenow fragment are more consistent with the elemental effect being the result of a steric clash in one of the transition states than with explanations based on electronegativity differences between sulfur and oxygen. The data suggest that the side chain of Asp882 is involved in the proposed steric clash, and that Gln849 and Glu883 participate in the sulfur-sensitive step of the reaction. Based on our results, and comparisons with other phosphoryl transfer enzymes, possible mechanisms for the polymerase reaction are discussed.

Amino Acids↗
PubMed1992

Social indicators and the prediction of psychiatric inpatient service utilisation.

The relationship between social variables and psychiatric service use in Victoria was investigated using a social indicators approach. Indicators were developed separately for the urban and rural areas of the state using 1986 census data. Principal components analysis was employed to reduce the data. Simple, unit weight indicators were developed and explained a considerable proportion of the variation in the rate of persons admitted to psychiatric facilities, the rate of admission episodes and the rate of occupied bed days for urban areas; a moderate amount of variation for these measures was accounted for by indicators developed for rural areas. The findings were considered in relation to both methodological issues and the role of other factors (e.g., accessibility and availability of psychiatric services) which may contribute to service use.

Adolescent↗
PubMed1991

Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-).

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

The 3'-5' exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction.

We have used site-directed mutagenesis to change amino acid side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease active site of Klenow fragment. Exonuclease assays of the resulting mutant proteins indicate that the largest effects on exonuclease activity result from mutations in a group of carboxylate side chains (Asp355, Asp424 and Asp501) anchoring two divalent metal ions that are essential for exonuclease activity. Another carboxylate (Glu357) within this cluster seems to be less important as a metal ligand, but may play a separate role in catalysis of the exonuclease reaction. A second group of residues (Leu361, Phe473 and Tyr497), located around the terminal base and ribose positions, plays a secondary role, ensuring correct positioning of the substrate in the active site and perhaps also facilitating melting of a duplex DNA substrate by interacting with the frayed 3' terminus. The pH-dependence of the 3'-5' exonuclease reaction is consistent with a mechanism in which nucleophilic attack on the terminal phosphodiester bond is initiated by a hydroxide ion coordinated to one of the enzyme-bound metal ions.

Amino Acid Sequence↗
PubMed1990

Identification of residues critical for the polymerase activity of the Klenow fragment of DNA polymerase I from Escherichia coli.

The Klenow fragment structure, together with many biochemical experiments, has suggested a region of the protein that may contain the polymerase active site. We have changed 7 amino acid residues within this region by site-directed mutagenesis, yielding 12 mutant proteins which have been purified and analyzed in vitro. The results of steady-state kinetic determinations of Km(dNTP) and kcat for the polymerase reaction, together with measurements of DNA binding affinity, suggest strongly that this study has succeeded in targeting important active site residues. Moreover, the in vitro data allow dissection of the proposed active site region into two clusters of residues that are spatially, as well as functionally, fairly distinct. Mutations in Tyr766, Arg841, and Asn845 cause an increase in Km(dNTP), suggesting that contacts with the incoming dNTP are made in this region. Mutations in the second cluster of residues, Gln849, Arg668, and Asp882, cause a large decrease in kcat, suggesting a role for these residues in catalysis of the polymerase reaction. The DNA-binding properties of mutations at positions 849 and 668 may indicate that the catalytic role of these side chains is associated with their interaction with the DNA substrate. Screening of the mutations in vivo for the classical polA-defective phenotype (sensitivity to DNA damage) demonstrated that a genetic screen of this type may be a reasonable predictor or kcat or of DNA binding affinity in future mutational studies.

Amino Acid Sequence↗
PubMed1990

The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.

The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity.

Base Sequence↗
PubMed1989

How DNA travels between the separate polymerase and 3'-5'-exonuclease sites of DNA polymerase I (Klenow fragment).

The polymerase and 3'-5'-exonuclease activities of the Klenow fragment of DNA polymerase I are located on separate structural domains of the protein, separated by about 30 A. To determine whether a DNA primer terminus can move from one active site to the other without dissociation of the enzyme-DNA complex, we carried out reactions on a labeled DNA substrate in the presence of a large excess of unlabeled DNA, to limit observations to a single enzyme-DNA encounter. The results indicated that while Klenow fragment is capable of intramolecular shuttling of a DNA substrate between the two catalytic sites, the intermolecular pathway involving enzyme-DNA dissociation can also be used. Thus, there is nothing in the protein structure or the reaction mechanism that dictates a particular means of moving the DNA substrate. Instead, the use of the intermolecular or the intramolecular pathway is determined by the competition between the polymerase or exonuclease reaction and DNA dissociation. When the substrate has a mispaired primer terminus, DNA dissociation seems generally more rapid than exonucleolytic digestion. Thus, Klenow fragment edits its own polymerase errors by a predominantly intermolecular process, involving dissociation of the enzyme-DNA complex and reassociation of the DNA with the exonuclease site of a second molecule of Klenow fragment.

Base Sequence↗
PubMed1988

Genetic and crystallographic studies of the 3',5'-exonucleolytic site of DNA polymerase I.

Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3',5'-exonuclease activity but having normal polymerase activity. Crystallographic analysis of the mutant proteins showed that neither had any alteration in protein structure other than the expected changes at the mutation sites. These results confirmed the presumed location of the exonuclease active site on the small domain of Klenow fragment and its physical separation from the polymerase active site. An anomalous scattering difference Fourier of a complex of the wild-type enzyme with divalent manganese ion and deoxythymidine monophosphate showed that the exonuclease active site has binding sites for two divalent metal ions. The properties of the mutant proteins suggest that one metal ion plays a role in substrate binding while the other is involved in catalysis of the exonuclease reaction.

Binding Sites↗
PubMed1987

Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli.

DNA polymerase I (Klenow fragment) of Escherichia coli catalyzes the addition of deoxynucleotides to 3' hydroxyl termini of blunt-ended DNA fragments. The product of the reaction, which we call +1 addition, is found only in very low yield under conditions that permit editing by the 3'----5' exonuclease activity of the wild-type polymerase. A mutant form of the Klenow fragment that lacks detectable 3'----5' exonuclease activity shows an elevated accumulation of the +1 addition product. The mutant enzyme can use any one of the four dNTPs to carry out the reaction when each precursor is provided individually. However, in the presence of all four dNTPs the addition of dATP is strongly preferred. Suppression of the editing function of the wild-type polymerase through the use of high concentrations of exogenous deoxynucleoside monophosphates also results in a significant increase in the amount of +1 addition product formed. The presence of a high dNMP concentration also alters the specificity of the nucleotide addition reaction carried out by the wild-type enzyme. Thus, in addition to dATP, the dNTP which is complementary to the exogenous deoxynucleoside monophosphate, is also used in the +1 addition reaction. A similar effect of dNMPs on the specificity of nucleotide addition was obtained with the mutant Klenow fragment. These results define two pathways for the +1 addition reaction: one that does not require coding information from the DNA template and a second in which coding information is provided by the exogenous dNMP.

Base Sequence↗