DNA Replication Fork: Pausing, Recombination and Disease

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DESCRIPTION (provided by applicant): Patients with tuberous sclerosis complex and autosomal dominant polycystic kidney disease most often are born with anatomically normal kidneys but develop significant renal involvement as they age. The abnormal tissues in these diseases are associated with the loss of heterozygosity (LOH) such that only the defective allele is present at the disease locus. Although both diseases have a second associated gene, the TSC2 and PKD1 genes cause a more severe phenotype and are more often found in patients with new mutations. We postulate that the disease severity is related to the fact that these adjacent genes are in an unstable region of chromosome 16. We present evidence that inverted Alu repeats and polypurine.polypyrimidine tracts from these genes appear to be associated with deletions, and block the human replication fork. In addition, the polypurine.polypyrimidine tract, under conditions that favor alternative secondary structure formation, can spontaneously initiate replication. Such replication initiation and termination phenomenon have both developmental and mutagenic implications. The ultimate goal of our reseach is to retard disease onset and progression by delaying the second somatic mutation leading to the LOH in the TSC2 and PKD1 genes. We hypothesize that alternative DNA secondary structures in the TSC2 and PKD1 genes promote mutagenesis through their effects on DNA replication. The proposed studies investigate the DNA structural characteristics of the Pu.Py tracts and inverted Alu repeats using 2-dimensional gel and melting curve analyses. Using repair deficient cell lines, we will also determine the replication proteins involved. Using a stable-transfection system, we will measure the ability of the sequences to stall replication and to induce recombination. The potential to initiate the human replication fork will also be studied in a well characterized system. By understanding the effects of these sequences on the fidelity of DNA replication, therapeutic inroads into delaying disease onset can be made.