Guanine-quadruplex DNA and polycystic kidney disease - 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.
