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-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.
Xue
Yu
Y
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Ratcliff
Glenn C
GC
Wang
Hong
H
Davis-Searles
Paula R
PR
Gray
Matthew D
MD
Erie
Dorothy A
DA
Redinbo
Matthew R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000071657:Werner Syndrome Helicase
D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D004247:DNA; D004720:Endonucleases; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase