Werner syndrome (WS) is a inherited recessive disease characterized by genetic instability. The disease is manifested clinically by premature aging and a high frequency of specific types of cancer. The identification and cloning of the WS gene offers the possibility of determining how it functions and how mutations in the gene are associated with genomic instability and cancer. On the basis of sequence homology, the WS gene was predicted to encode a DNA helicase. The PI has inserted the gene into baculovirus DNA, transformed insect cells and over-expressed the WS protein. They have established that the recombinant protein unwinds double-stranded DNA in the 3'>5' direction. The production of active WS protein should allow the definition of its function by using in vitro model systems and allow further analysis of the role of mutant WS proteins in the generation of genetic instability. In order to further characterize the WS helicase activity, it is proposed to purify the wild type and mutant WS proteins to homogeneity. In vitro studies will be performed to 1) define substrate specificity, progressivity and stoichiometry, 2) identify interacting proteins and 3) assess the involvement of DNA repair, replication and recombination. These in vitro studies should provide significant clues to the function of WS proteins in cells. In addition, methods will be developed to identify carriers with only one mutant WS gene and determine if cells from these individuals are genetically unstable.
