Research Project
9656032
Project Summary In Autosomal Dominant Polycystic Kidney Disease a mutation in one PKD1 gene is inherited, but a somatic mutation in the second normal allele is required for cyst formation. The human PKD1 gene is unstable and susceptible to inactivating mutagenesis, resulting in the relatively high disease incidence of 1 in 500 individuals. The mechanisms responsible for PKD1 mutagenesis are not resolved. However, one important clue comes from sequence analysis; the human PKD1 gene is unusually repetitive. By comparison, the murine homolog is genetically stable and not particularly repetitive. This implies that an underlying source of mutagenesis for the human PKD1 gene is the sequence itself. Certain DNA repeat sequences in the genome are already known to be functionally associated with locus-specific recombination and mutagenesis. Tandem repeats of guanine, in particular, are concentrated at mutation hot spots where they support the formation of four-stranded DNAs called G-quadruplex (G4) DNA. At the immunoglobulin genes, G4 DNA is involved in the programmed rearrangements necessary for proper immune responses. At other genomic loci, G4 DNA increases the frequency of spontaneous rearrangements that result in diseases, such as cancer. Unresolved G4 DNAs are mutagenic because they present a barrier to polymerase progression during transcription or replication. We have discovered that the human PKD1 gene contains widespread G4 DNA sequence motifs. They are clustered together within specific introns, but also widely distributed throughout the gene. We hypothesize that G4 DNA structure formation within human PKD1 promotes somatic mutagenesis, leading to gene inactivation and then cyst formation. In order to test that model and uncover a mechanism for PKD1 inactivation, we will assay for the presence of G4 DNA structures in PKD1 in vitro and in vivo in three specific Aims. In Aim 1, we will use Circular Dichroism Spectroscopy (CD) and R-loop transcription assays to characterize G4 DNA and co-transcriptional structures formed from PKD1 sequence repeats. Aim 2 will directly test for G4 formation within PKD1 in the cell by Chromatin Immunoprecipitation (ChIP) using a G4 DNA-specific antibody. G4 DNA is known to promote oncogene translocations, so Aim 3 will use a yeast reporter assay to determine if DNA rearrangements occurring in PKD1 depend upon G4 formation. Outcomes of our studies will link, for the first time, the instability of the PKD1 gene with G4 DNA structures, revealing a mechanism of gene inactivation that explains why ADPKD occurs.
