In this competitive renewal application, we propose to study the mechanisms of genetic instability focusing on simple repeat sequences. A genetic instability that produces an expansion of trinucleotide repeats has been observed in several hereditary diseases. Frequent short deletions and insertions within (CA)n repeats were also found in hereditary colorectal cancers. The mechanism underlying trinucleotide expansion is unknown. While a defect in mismatch repair may well play a role in the mechanisms of the mutation, it is likely that structural property of simple repeat sequences is as equally important. Characterization of the intrinsic properties of repeated DNA and factors interacting with these DNA is essential for understanding the mechanism of genetic instability involving simple repeat sequences. We have shown that many simple repeat sequences have intrinsic properties to form unusual DNA (or non-B DNA) structures under superhelical strain in vitro and in vivo. Our results suggested that certain simple repeat sequences can regulate gene expression and recombination through adoption of non-B DNA structures. Two lines of research on the role of simple repeat sequences on genetic instability have developed during the current funding period. The first part of the research focuses on AGC trinucleotide repeat sequences. To understand the mechanisms of trinucleotide repeat sequence expansion, we hypothesized that trinucleotide repeats have a high potential to adopt unusual DNA structures. indeed, we found that the (AGC)n trinucleotide repeats form a novel non-B DNA structure in vitro. This unusual structure may interfere with replication and/or other processes involving repeated DNA, and specialized proteins might exist to deal with these potential problems. We have purified (AGC)n-binding proteins from mouse brain and have recently isolated a cDNA that encodes an (AGC)n-binding protein (mAGC-BP), predominantly expressed in the brain. The cDNA may serve as a tool to understand the mechanisms of DNA expansion mutations in genetic disorders. We propose in Specific Aim Part 1 to characterize the cDNA encoding the AGC-repeat sequence binding protein, and to study biological functions of the protein. The second part of the research is on simple repeat sequences playing a role in deletion mutation.
This aim i s based on our recent finding that poly(dG)-poly(dC) sequences are induced to form a dG.d6.dC intramolecular triplex structure formation in cells in response to transcriptionai activation of a downstream gene, and this triggers deletion mutations between direct repeat sequences flanking the dG tracts. This model system will allow us to study the effect of intrinsic properties of various simple repeat sequences on deletion mutation. We propose in Specific Aim Part II to further investigate the mechanism for this transcription- induced recombination and to study the roles of simple repeat sequences on deletion mutation upon transcriptional activation in E.coli cells.
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