This is a new application which seeks to investigate the specificity of potentially new types of mismatch endonucleases. The hypothesis is that the plant systems provide a good means for understanding mismatch repair and gene conversion and for carrying out genetics, and that mismatch-specific multi-purpose endonucleases in plants may be conserved in other eukaryotes including humans. Specifically, (1) a celery mismatch endonuclease CEL I, identified by the principal investigator, will be purified and characterized biochemically, together with celery DNA polymerases and DNA ligases to form a complete catalytic repair system. Other protein factors or co-factors that may participate in this reaction will be purified. The complete mismatch repair reaction will be reconstituted in vitro and characterized. (2) An immunological approach will be taken to dissect the mismatch endonuclease system. Antibodies to CEL I and its homologues will be made either as mouse hybridoma monoclonals or as phage-displayed recombinant antibodies, and used for studying the relationship of the nucleases from different organisms, for affinity purification of the enzymes, and for gene cloning. It is anticipated that the apparent conservation of these enzymes among plants should allow the principal investigator to apply these approaches to the Arabidopsis model system that is amenable to genetic analysis. (3) A molecular genetics approach is to be taken to understand the Arabidopsis thaliana mismatch endonuclease system. The antibody tools developed above should enable cloning and genetic studies in this excellent model system. The DNA sequence information will enable them to look for human homologues and determine whether yeast genetics can be employed.
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