Christopher Mathews
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Emeritus Distinguished Professor, Biochemistry and Biophysics |
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Education
Ph.D. 1962, University of WashingtonResearch
Our general interest is nucleic acid enzymology. We focus upon the biosynthesis of DNA precursors-- metabolic and genetic control, properties of the individual enzymes, and relationship to DNA replication, repair, and mutation. We work with bacterial and mammalian cells and viruses. Much of the interest in these enzymes pertains to their status as targets for antiviral and anticancer drugs.Infection of Escherichia coli by phage T4 elicits the synthesis of many enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis, as well as enzymes and proteins of DNA replication. The enzymes of dNTP synthesis interact to form a large supramolecular complex. This may help channel precursors to replication sites, and hence, to maintain high rates of DNA chain growth.
We have purified the complex and are studying its structure and its relationship to the replication apparatus. This work involves protein fractionation, immunological techniques, protein cross-linking, enzyme kinetics, affinity chromatography, and two-dimensional protein gel electrophoresis. Evidence suggests a model for the relationship between the dNTP synthetase complex and the replication fork, in which the complex associates with the single-strand DNA-binding protein encoded by gene 32.
A related project concerns the regulation of ribonucleotide reductase, which catalyzes the first committed reaction in DNA synthesis. We have devised an assay that simultaneously monitors the four activities of this enzyme (reduction of ADP, CDP, GDP, and UDP). We have learned that intracellular substrate concentrations are critical determinants of specificity in vivo and are currently trying to identify how other perturbations--oxygen limitation, free-radical trappers, and purine nucleotide imbalances--affect intracellular activities. Also, we have found that mammalian mitochondria contain a distinctive form of ribonucleotide reductase. By analyzing dNTP pools in mitochondria from rat tissues, we have found surprising asymmetries. In some tissues, dGTP comprises 90 percent of the total dNTP and dTTP is almost undetectable. In vitro experiments with mitochondrial DNA polymerase indicate that this asymmetry probably contributes to the high mutation rate of the mitochondrial genome.
We are investigating dNTPs as targets for mutagenesis. Particular interest focuses upon oxidizing agents that lead to the oxidized DNA base, 8-oxoguanine, which base-pairs with adenine and leads to a tranversion mutation. Evidence suggests that much of the biologically relevant oxidation occurs at the nucleotide level, and we have developed an HPLC assay to measure pool sizes of the oxidized nucleotide, 8-oxo-dGTP, in cell extracts. Although we find dGTP to be the least abundant dNTP in mammalian or bacterial cells (unlike what we see in mitochondria) our data call into question the widespread belief that dGTP oxidation in vivo is a significant contributor to mutagenesis induced by reactive oxygen species.