Cells employ numerous DNA repair and mutation avoidance mechanisms to protect genetic integrity. In the absence of these important processes, cells suffer mutations, chromosomal aberrations or death. Repair-deficient human syndromes have been identified and include neurological and immunological defects, cancer-proneness and premature aging. One common feature of many DNA repair and mutation avoidance mechanisms is the degradation of DNA, accomplished by DNA exonuclease proteins. Exonucleases excise offending DNA lesions or replication errors and promote recombinational repair of broken chromosomes. Exonucleases also prevent inappropriate genetic rearrangements that lead to mutation. Exonucleases produce molecular signals for cell division arrest when the cell is confronted with DNA damaged. A molecular understanding of DNA recombination, repair and mutagenesis will require knowledge of the exonucleases that participate in these processes. Our objective is to define recombination and repair exonucleases of E. coli and Saccharomyces cereviseae. We seek to understand their biochemical properties, their molecular partners and what roles they play in vivo. The RecJ exonuclease from E. coli has been the focus of much of our previous investigation. We have shown that RecJ is a member of a large family of proteins found in archaebacteria, eubacteria and eukaryotes. We will continue to analyze the structure and function of this protein. Physical or functional interactions of RecJ exonuclease with other proteins involved in DNA replication or repair will be assayed. We have identified two new exonucleases from the bacterium E. coli. We will continue to characterize their biochemistry and will analyze mutants in these exonucleases for genetic stability, recombination and DNA repair defects. As it is clear that some of these functions are genetically redundant, multiple mutants in these and other genes will assessed for genetic properties. Physical monitoring of DNA repair and assessment of SOS regulation will be performed in ssExo mutants. A third putative DNA exonuclease from E. coli will be assayed for activity on oligonucleotides. Mutants and genetic suppressors of this function will be characterized. We will investigate the role of putative 3' exonucleases (based on sequence similarity) from the yeast Saccharomyces cerevisiae. The genes will be expressed in E. coli to verify if they encode exonucleases. Mutants in conservied residues will be examined for biological effects.
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