This proposal has two long goals: the first goal is to understand the enzymatic mechanism(s) by which mismatched nucleotides are repaired in the simple eukaryote Saccharomyces cerevisiae and the second goal is to use mismatch repair enzymes and monoclonal antibodies that recognize mismatched nucleotides to develop methods for mapping the position of mutations in eukaryotic genes. An already developed in vitro system that uses extracts of mitotic S. cerevisiae cells to catalyze the repair of mismatched nucleotides will be studied in detail to identify individual steps in the repair reaction. The extracts will be fractionated to identify and purify individual proteins that are required for mismatch repair. Each protein will be characterized in detail to identify any enzymatic activities it has and to determine the role it plays in the mismatch repair reaction. The genes that encode each protein will be cloned in order to both overproduce each protein and to carry out genetic experiments to determine the in vivo role of each protein. The ultimate goal of these first experiments will be to reconstitute mismatch repair reactions with purified proteins and defined substrates and determine the enzymatic mechanism(s) of these reactions. An already identified endonuclease activity that makes double-strand breaks at the site of single mispaired bases will be purified to homogeneity and characterized to determine its cleavage specificity. The gene encoding the endonuclease will be cloned in order to both overproduce the endonuclease and to carry our genetic experiments to determine if the endonuclease plays a role in repairing replication errors and other mismatch repair reactions as postulated by recent models. The endonuclease will then be used to develop methods for mapping the position by recent models. The endonuclease will the be used to develop methods for mapping the position of mutations in genes by forming heteroduplexes between wild type and mutant DNAs and then mapping the position of the resulting mispaired nucleotides by cleaving them with the endonuclease and mapping the position of the double- strand break. Cloned mutant Human beta-globin genes and wild type chromosomal DNA will be used as a test system for the mapping experiments. Monoclonal antibodies that specifically recognize individual mispaired nucleotides will be developed and characterized. These mispair-specific antibodies will be used to extend the mapping procedure so that it will be possible to identify the exact base change that is present at a mismatched site. These latter experiments should lead to identify the exact base change that is present at a mismatched site. These latter experiments should lead to the development of simple methods for carrying out fine structure gene mapping in eukaryotes.
