Effects of Denys-Drash syndrome point mutations on the DNA binding activity of the Wilms' tumor suppressor protein WT1

A number of point mutations in the zinc finger domain of the Wilms' tumor suppressor protein WT1 have been isolated from the DNA of patients with Denys-Drash syndrome, an association of Wilms tumor, nephropathy, and genital anomalies, To date, five different mutations that alter amino acids predicted to interact specifically with nucleotides in the target DNA sequence have been described. Two of these mutations are located in zinc finger 2 (R366H, R366C), and three are located in finger 3 (R394W, D396G, D396N). These five Denys-Drash mutations were introduced into WT1-ZFP, a recombinant polypeptide containing rhs zinc finger domain of WT1, and the effects of these mutations on DNA Sequence specificity were determined using a selection, amplification, and binding (SAAB) assay. The SAAB assay was carried out using two different DNA templates, one with a randomized finger 2 subsite (GCG TGG NNN TGT) and one with a randomized finger 3 subsite (GCG NNN GCG TGT). A comparison of the DNA sequences selected by WT1-ZFP and by Denys-Drash mutants suggests that the point mutations reduce the sequence selectivity of the zinc finger protein. With the exception of the R394W mutant, the other Denys-Drash mutations selected one alternative sequence in addition to the wild-type DNA subsite sequence. The binding affinities of these proteins for their selected sequences were determined using a quantitative nitrocellulose filter binding: assay. These results revealed that the wild-type WT1 binds with slightly higher affinity to sequences with GAG in the finger 2 subsite than sequences with the EGR-1 consensus GCG finger 2 subsite. With the exception of R394W, which appears to lack specific DNA binding activity, the Denys-Drash mutants bound to selected DNAs with 1.4-14-fold lower affinities than the wild-type WT1-ZFP. These results suggest that the clinical phenotype of Denys-Drash syndrome can be associated with a modest reduction in the DNA binding affinity of WT1.