The biological effects of Pu·Py tracts remain to be fully elucidated. The studies presented here demonstrate that mirror repeat Pu·Py tracts from PKD1
intron 21 form DNA intramolecular triplex structures under modest energies and inhibit replication. Bacolla et al
. found that nonB-DNA structures formed by the PKD1
Pu·Py tract inhibited replication in Escherichia coli
). We demonstrate that these tracts inhibit replication in eukaryotic replication systems as well. Such interference with DNA replication can lead to mutation (40
We thermodynamically characterized intramolecular triplex formation by the Pu·Py mirror repeats found at the P1 nuclease-sensitive regions of PKD1
intron 21. Blaszak (24
) found that pJB4b formed a triplex at σ = –0.0375. pJB4b is the longest individual Pu·Py mirror repeat from the region of intron 21 examined. We found that shorter Pu·Py mirror repeats from this region also form triplexes at modest superhelical tension. Sequences containing overlapping Pu·Py tracts required significantly less energy to form triplexes than individual Pu·Py tracts. This observation may indicate that the longer repetitive tracts undergo slipped mispairing more frequently, lowering the energy required to melt the sequence and making transition to a triplex structure more likely.
Although the physiologic relevance of the ability of PKD1 Pu·Py sequences to adopt an alternative secondary structure in vitro under acidic conditions is unclear, it does appear to predict replication arrest. The replication fork blockade demonstrated in Figures D and A most likely represent replication arrest due to a Pu·Pu·Py triplex formation between the nascent and template strands during replication with the polypurine template strand folding back over the polypyrimidine nascent strand. This interstrand effect on replication would not be dependent on superhelical tension given the single-stranded template. While independent of superhelical tension, however, the foldback-triplex formation would be expected to share common features with the intramolecular triplex formation. For example, the energy required for foldback-triplex formation may correlate with the superhelical energy required for intramolecular triplex formation. Additionally, a longer mirror repeat sequence would be more likely to form either kind of triplex and would probably be more difficult for the polymerase to bypass.
We looked for other features of the Pu·Py tracts that correlate with polymerization arrest in an effort to identify sequence characteristics that can be used to predict which Pu·Py tracts in the human genome are more likely to interfere with replication. Besides superhelical tension of intramolecular triplex formation, we found that only the potential length of the triplex had a statistically significant correlation with polymerization arrest. The relationship between the length of the Pu·Py tracts and polymerase pausing has been suggested previously (28
). As shown in Figure E, the primer extension studies demonstrated a considerable range in the amount of polymerase blockade. Given the correlation coefficients obtained, this variability is only partially explained by the factors examined in this study, reflecting the role of additional factors.
A limitation of this study is that Taq polymerase was used, which is not as processive as other polymerases. This limitation in processivity may explain the multiple bands observed in the primer extension studies that were not consistent with either a full-length product or polymerase arrest within the Pu·Py region. Because repeat sequences are prone to slipped mispairing mutagenesis, digestion of the plasmids with EcoRI was performed and showed a single length insert size, indicating that the starting templates for the primer extensions were homogenous in size (see Fig. S1).
Another limitation is that replication blockade using purified Taq polymerase does not model eukaryotic replication systems. We therefore examined the effect of the PKD1 Pu·Py tracts on eukaryotic replication systems using both a primer extension assay (Fig. A) and and an SV40 system (Fig. C). Both assays demonstrated significant replication blockade in the presence of a Pu·Py tract from PKD1 (Fig. A and C). In addition, the SV40 replication assay shows the presence of double-strand breaks which is only observed in the presence of the Pu·Py tract (Fig. C). Both findings further support the hypothesis that these sequences play a role in the development of human ADPKD.
A disease model is proposed in which the G-rich template of Pu·Py mirror repeat tracts of PKD1
or its homologous genes engage in a foldback-triplex structure with the nascent strand during DNA replication resulting in a replication fork blockade (Fig. ). In an effort to reactivate the blocked replication fork, recombination or double-strand breaks may be required which can compromise replication fidelity, leading to a second hit mutation in the normal PKD1
allele and LOH. Such a model is consistent with the gene conversion mutagenic mechanism proposed by Watnick et al
). Following replication blockade, gene conversion can occur using the homologous tracts of the PKD1
gene at chromosome 16p13.1 (42
Figure 5 Flow diagram of the disease model. The replication blocking effect of the mirror repeat Pu·Py tract is a probability. A small percentage of the replication fork will be blocked. The majority of blocked forks are reactivated in a mutation-free (more ...)
This model supports the potential role of the Pu·Py tract in PKD1 or homologous genes as a mechanism leading to somatic mutation and cystogenesis in ADPKD. Variation in the rate at which this second hit occurs may explain the variable expressivity seen with ADPKD. The development of the ability to predict which Pu·Py tracts are more likely to lead to mutagenesis and the development of methods to prevent or slow down the rate of triplex formation during replication may offer an opportunity to alter the disease course in these patients for whom only supportive treatment is currently available.