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Biomedical subjects

M P Fitzgerald

Publications and source records attributed to M P Fitzgerald.

9 recordsLinked to original sources
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↗
PubMed1990

Fidelity of animal cell DNA polymerases alpha and delta and of a human DNA replication complex.

We are investigating the mechanisms by which mutations are produced or avoided during DNA synthesis. Using in vitro fidelity assays, we have defined the error frequency and mutational specificity of the replicative animal cell DNA polymerases (alpha and delta). With DNA polymerase alpha or the four-subunit DNA polymerase alpha-DNA primase complex, neither of which contains detectable associated exonuclease activity, the fidelity of the polymerization step is low relative to spontaneous mutation rates in vivo. DNA polymerase delta is much more accurate, partly due to proofreading by the 3'----5' exonuclease activity associated with this polymerase. These fidelity studies have been extended to the replication apparatus present in extracts of human HeLa cells. The replication complex is highly accurate, suggesting that additional fidelity components are operating in the extract during bidirectional, semiconservative replication of double-stranded DNA. Nevertheless, in highly sensitive reversion assays, base substitution errors can be readily detected at frequencies greater than the estimated rate of spontaneous mutation in vivo. This suggests that fidelity components may be missing and/or that human cells depend heavily on postreplicative repair processes to correct replication errors.

Animals↗
PubMed1990

Evaluation of the bioluminescence assays as screens for genotoxic chemicals.

While present data suggest that the Mutatox assay is not effective for carcinogen screening, it does appear to detect many agents with diverse mechanisms of mutagenic activity with reasonable sensitivity as well as some known human teratogens. For general screening, more detailed studies need to be performed using coded agents with various mutagenic mechanisms as well as with toxicants of various classes including teratogens. With additional validation, the Mutatox assay, with its sensitivity to diverse chemical classes, may be valuable as a rapid, short-term, cost-effective assay.

Carcinogenicity Tests↗
PubMed1989

Fidelity of DNA polymerase I and the DNA polymerase I-DNA primase complex from Saccharomyces cerevisiae.

We have determined the fidelity of DNA synthesis by DNA polymerase I (yPol I) from Saccharomyces cerevisiae. To determine whether subunits other than the polymerase catalytic subunit influence fidelity, we measured the accuracy of yPol I purified by conventional procedures, which yields DNA polymerase with a partially proteolyzed catalytic subunit and no associated primase activity, and that of yPol I purified by immunoaffinity chromatography, which yields polymerase having a single high-molecular-weight species of the catalytic subunit, as well as three additional polypeptides and DNA primase activity. In assays that score polymerase errors within the lacZ alpha-complementation gene in M13mp2 DNA, yPol I and the yPol I-primase complex produced single-base substitutions, single-base frameshifts, and larger deletions. For specific errors and template positions, the two forms of polymerase exhibited differences in fidelity that could be as large as 10-fold. Nevertheless, results for the overall error frequency and the spectrum of errors suggest that the yPol I-DNA primase complex is not highly accurate and that, just as for the polymerase alone, its fidelity is not sufficient to account for a low spontaneous mutation rate in vivo. The specificity data also suggest models to explain -1 base frameshifts in nonrepeated sequences and certain complex deletions by a direct repeat mechanism involving aberrant loop-back synthesis.

Base Sequence↗
PubMed1976

Photosynthesis in a reconstituted chloroplast system from spinach. Some factors affecting CO2-dependent oxygen evolution with fructose-1,6-bisphosphate as substrate.

When envelope-free spinach chloroplasts are incubated with stromal protein, catalytic NADP, catalytic ADP, radioactive bicarbonate and fructose 1,6-bisphosphate, 14CO2 fixation starts immediately upon illumination but oxygen evolution is delayed. The delay is increased by the addition of fructose 6-phosphate and by a variety of factors known (or believed) to increase fructose bisphosphatase activity (such as dithiothreitol, more alkaline pH, higher [Mg] and antimycin A). Conversely, the lag can be decreased or eliminated by the addition of an ATP-generating system. Bearing in mind the known inhibition, by ADP, of sn-phospho-3-glycerate (3-phosphoglycerate) reduction it is concluded that the lag in O2 evolution results from the production of ribulose 5-phosphate from fructose bisphosphate and that this in turn inhibits the reoxidation of NADPH by adversely affecting the ADP/ATP ratio. The results are discussed in their relation to the mode of action of antimycin A and to regulation of the reductive pentose phosphate pathway.

Carbon Dioxide↗