Abstract
Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb.
Highlights
Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation*
Because Ku and DNA-PKcs are subunits of DNAPK, which is involved in the repair of DNA dsb, we explored the possibility that Werner protein (WRN) exonuclease activity is modulated by DNA-PKcs
Because Ku functionally modulates the activities of both WRN and DNA-PKcs, we assayed for the modulation of WRN exonuclease activity by DNAdependent protein kinase (DNA-PK)
Summary
Vol 277, No 21, Issue of May 24, pp. 18291–18302, 2002 Printed in U.S.A. Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation*. WS cells exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks [12, 13] These studies suggest that WRN plays an important role in DNA metabolism possibly by participating in DNA repair, replication, and/or recombination pathways. We have previously shown that Ku significantly stimulates the 3Ј35Ј exonuclease activity of WRN [14], suggesting that WRN and Ku could act in a common pathway of DNA metabolism involving the exonuclease function of WRN This hypothesis is supported by the observation that mice lacking the Ku80 subunit show a premature aging phenotype similar to that of WS patients [17]. Our results would suggest that WRN may play a role in DNA-PK-mediated end rejoining
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