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' tu mor 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 l ocated in zinc finger 2 (R366H, R366C), and three are located in finge r 3 (R394W, D396G, D396N). These five Denys-Drash mutations were intro duced into WT1-ZFP, a recombinant polypeptide containing rhs zinc fing er domain of WT1, and the effects of these mutations on DNA Sequence s pecificity were determined using a selection, amplification, and bindi ng (SAAB) assay. The SAAB assay was carried out using two different DN A 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 compar ison of the DNA sequences selected by WT1-ZFP and by Denys-Drash mutan ts suggests that the point mutations reduce the sequence selectivity o f the zinc finger protein. With the exception of the R394W mutant, the other Denys-Drash mutations selected one alternative sequence in addi tion to the wild-type DNA subsite sequence. The binding affinities of these proteins for their selected sequences were determined using a qu antitative nitrocellulose filter binding: assay. These results reveale d that the wild-type WT1 binds with slightly higher affinity to sequen ces with GAG in the finger 2 subsite than sequences with the EGR-1 con sensus GCG finger 2 subsite. With the exception of R394W, which appear s 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-D rash syndrome can be associated with a modest reduction in the DNA bin ding affinity of WT1.