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

Biochemistry. 2002 Mar 5;41(9):2901-12. doi: 10.1021/bi0157161.

Abstract

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-N(70-240)), 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-N(70-240) 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-N(70-240) forms a hexamer and interacts with the recessed 3'-end of the DNA. Moreover, we find that the interaction of hWRN-N(70-240) 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Chromatography, Gel
  • Cloning, Molecular
  • DNA / metabolism*
  • DNA Helicases / chemistry
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • Endonucleases / metabolism*
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism*
  • Humans
  • Mice
  • Molecular Sequence Data
  • Proliferating Cell Nuclear Antigen / metabolism
  • Protein Structure, Tertiary
  • RecQ Helicases
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Werner Syndrome Helicase

Substances

  • Proliferating Cell Nuclear Antigen
  • Recombinant Proteins
  • DNA
  • Endonucleases
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V
  • DNA Helicases
  • RecQ Helicases
  • WRN protein, human
  • Werner Syndrome Helicase