Mutations associated with imperfect inverted repeat sequences, """"""""quasipalindromes"""""""", with potential for DNA secondary structure, are widespread. They have been noted as mutational hotspots in bacteriophage, yeast and humans and mutate by a replication template-switch mechanism. In humans, template-switch mutations contribute to a large set of genetic diseases, structural variation in genomes and to 20% of the mutations that affect p53 in human cancers. Despite the prevalence and importance of this class of mutation, little systematic work has been done to define the parameters that govern the mutagenic mechanism, mutagens specific to this class or cellular factors that influence mutation rate. The long-term goal of this study is a more complete mechanistic understanding of quasipalindrome-associated mutagenesis. Objectives will be to define the structural parameters and cellular pathways that govern mutability. Because all cells mutate and repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organisms, Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster should reveal mechanisms applicable to repair of DNA in human cells.
The first aim of this proposal is to investigate the connection between replication, DNA damage repair and transcription collisions with template-switch mutagenesis in E. coli. The hypothesis that targeting of exonucleases to lagging strand features via singlestrand DNA binding protein and replication clamps accounts for the observed strand bias of mutagenesis will be tested. The impact of replication fork collisions with transcription complexes will be assessed. Biochemical analysis of DNA polymerase III and its interactions with clamp and clamp-loader will be correlated with genetic effects on mutagenesis to clarify its mechanism. Additional template-switch vulnerable sites will be sought by whole-genome sequence analysis.
The second aim i s to develop the first eukaryotic mutational reporters for template-switch mutagenesis using the URA3 gene of S. cerevisiae. In addition, an existing lacZ reporter for mutagenesis in Drosophila melanogaster will be retrofitted to report specific types of mutations, including template-switching at quasipalindromes or direct repeats, and base substitutions and frameshifts that respond to particular types of polymerase errors or DNA damage. The effects of aging on mutation frequency will be tested using these constructs. This study will significantly advance our understanding of DNA mutagenesis by providing new tools and information about this important and neglected class of mutations.
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