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John Hays
Professor, Environmental and Molecular Toxicology
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RESEARCH:
Genetic Fidelity Mechanisms. Cells protect the genetic integrity of their genomes, which in eukaryotes can exceed 109 base-pairs, with astonishing success. Critical to the low error rate for DNA replication (10-10 - 10-9 mistakes per generation per bp) are highly-conserved mismatch-repair systems. Ultraviolet-light impact on the biosphere is expected to increase as long as stratospheric ozone depletion increases solar UV-B intensities. Cells resist the cytotoxic, mutagenic and carcinogenic consequences of UV-damaged DNA by an ensemble of repair and damage-toleration mechanisms.
Genetic Fidelity and Somatic Mutation in Plants. Since plants lack a reserved germ line, they must minimize spontaneous and environmental mutagenesis, particularly in the somatic meristematic cells that give rise to flowers and seeds. To elucidate the roles of plant mismatch repair systems in genetic fidelity, we have isolated six mmp genes of the small "model" green plant, Arabidopsis thaliana, and are characterizing their gene products. As in human and other eukaryotic cells, atMSH2 forms heterodimers with atMSH3 and atMSH6 proteins, respectively; these have different mismatch-binding specificities. Current focus is on the roles of atMSH2 and atMSH7, both of which may be targeted to the mitochondrion. The effects of overexpression in transgenic Arabidopsis of expected dominant-negative atMSh2, atMLH1, and atPMS2 mutant genes on mutation of microsatellite sequences and transgenes encoding Green Fluorescon Protein and on meiotic recombination and fertility, is under study.
Antagonism of UV and Alkylation Mutagenesis in Human and Bacterial Cells by Mismatch Repair. The recognition components of mismatch-repair (MMR) systems show extremely high conservation of critical domains during their evolution from single bacterial MutS proteins to a set of specialized eukaryotic MutS homologs (MsHs). Besides recognizing and correcting DNA replication errors, mismatch-repair systems process DNA containing certain damaged bases MutS/MSH proteins bind specifically to DNA containing "mismatched" UV photoproducts, e.g. T<>T/AG but not "matched" T<>T/AA, and mismatch repair reduces UV mutagenesis and perhaps UV-induced skin cancer. The specificity, efficiency, and accuracy of mismatch repair of UV-damaged DNA is being analyzed in human and bacterial cell extracts. DNA containing highly mutagenic 106-methylguanine adducts, introduced by certain anti-tumor drugs, is also processed by mismatch repair. We are analyzing the biochemical mechanisms involved .
Replication and Repair of Nuclear, Chloroplast, and Mitochondrial DNA in UV-irradiated Plants. We are using polymerase-chain reaction (PCR) primers specific for Arabidopsis nuclear (nc), chloroplast (cp), and mitochondrial (mt) genomes to measure their replication during plant development and after UVB irradiation. Replication of all three genomes shows a remarkable ability to recover from high levels of UV photoproducts (which we measure by immuno-assays). We are following the time courses of photoproduct retention/disappearance in irradiated nc, cp, and mt DNA, to distinguish repair from mechanisms of damage toleration, such as recombination.