A minimal exonuclease domain of WRN forms a hexamer on DNA and possesses both 3′-5′ exonuclease and 5′-protruding strand endonuclease activities

Werner syndrome is a rare autosomal recessive disease characterized by a premature aging phenotype. genomic instability, and a dramatically increased incidence of cancer and heart disease. Mutations in a single gene encoding a 1432-amino acid helicase/exonuclease (hWRN) have been shown to be responsible for the development of this disease. We have cloned, overexpressed, and purified a minimal, 171-amino acid fragment of hWRN that functions as an exonuclease. This fragment, encompassing residues 70-240 of hWRN (hWRN-N70-240), exhibits the same level of 3'-5' exonuclease activity as the previously described exonuclease fragment encompassing residues 1-333 of the full-length protein. The fragment also contains a 5'-protruding DNA strand endonuclease activity at a single-strand-double-strand DNA junction and within single-stranded DNA, as well as a 3'-5' exonuclease activity on single-stranded DNA. We find hWRN-N70-240 is in a trimer-hexamer equilibrium in the absence of DNA when examined by gel filtration chromatography and atomic force microscopy. Upon addition of DNA substrate, hWRN-N70-240 forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N70-240 with the replication protein PCNA also causes this minimal, 171-amino acid exonuclease region to form a hexamer. Thus, the active form of this minimal exonuclease fragment of human WRN appears to be a hexamer. The implications these results have on our understanding of hWRN's roles in DNA replication and repair are discussed.