Increased levels of spontaneous mutations have been associated with many human genetic diseases. The cellular processes that influence spontaneous damage and the spontaneous mutation rate however, are largely unknown. Metabolic by-products are a significant factor contributing to spontaneous mutagenesis, and perhaps the most predominant lesions generated by endogenously-produced agents are AP sites. AP endonucleases constitute a set of enzymes that initiate repair of potentially lethal and/or mutagenic 3'- blocking lesions and AP sites in DNA. The proposed research will examine the role of the major yeast Saccharomyces cerevisiae AP endonuclease (Apn1) in the control of spontaneous mutations in a variety of human target sequences. Human mutational target sequences will be installed in the yeast genome by integrative recombination. The integrating vectors will contain the target sequence inserted in several orientations relative to the promoter and replication direction so that we will be able to assess whether endogenously induced AP sites are repaired in a replication- or transcription-coupled fashion. These studies will help us understand how specific cellular processes such as repair (Apn1), transcription, and replication influence spontaneous mutagenesis.
