Werner Protein Is a Target of DNA-dependent Protein Kinase in Vivo and in Vitro, and Its Catalytic Activities Are Regulated by Phosphorylation

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 335 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 DNAdependent 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. Werner syndrome (WS)1 is a human autosomal recessive disorder characterized by early onset of premature aging characteristics including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. In addition, WS patients exhibit an increased incidence of diabetes mellitus type 2, hypertension, and malignancies (1). The gene (WRN), defects in which are responsible for WS, encodes a 1,432-amino acid protein (WRN) (2) that has both 3Ј35Ј helicase and 3Ј35Ј exonuclease activities (3-7). Although WRN appears to play an important role in DNA metabolism, the precise cellular roles of both the helicase and exonuclease activities of WRN remain to be determined.Cells from patients with WS show premature replicative senesence compared with cells derived from normal individuals (8). The WS cellular phenotype suggests correlations among faulty DNA metabolism, genomic instability, and senescence. WS cells show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO) (9), topoisomerase inhibitors (10), and certain DNA cross-linking agents (11). Compared with normal cells, WS cells also 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, re...

Cancer and aging: the importance of telomeres in genome maintenance

“…TRF2 also interacts with the DNA damage sensing protein ATM, and is thought to inhibit ATM activity specifically at telomeres [81], and WRN [82], the protein that is defective in the human premature aging and cancer-prone disorder Werner syndrome [83,84]. WRN encodes a DNA helicase and exonuclease [85,86] that appears to participate in both the NHEJ and HR DNA repair pathways [87][88][89][90][91][92][93][94]. It is not known whether all TRF1 complexes contain TANK1/2 and/or Ku, or whether all TRF2 complexes contain ATM, WRN, the RMN complex and/or other DNA damage sensors or repair proteins (Fig.…”

Deficient DNA repair in the human progeroid disorder, Werner syndrome

“…Ku is part of the DNA-PK complex, and associates with DNA and the DNA-PK catalytic subunit to form an active kinase [19]. DNA-PK-dependent phosphorylation of WRN has been observed in vitro and in vivo, and regulates the WRN helicase and exonuclease activities [22,23]. Aberrant products of NHEJ reactions have been observed in WS cells [24].…”

DNA Repair and Tumorigenesis: Lessons from Hereditary Cancer Syndromes

“…84 Phosphorylation of WRN by the ATM/ATR-related kinase DNA-PK inhibits its exonuclease activity; suggesting that DNA-PK may regulate the different activities of WRN. 88 The WRN helicase function appears active on multiple DNA replication/repair intermediates such as a 5´-ssDNA flap and a three-strand junction. 89 These latter results are consistent with a role for WRN in resuming a stalled replication fork.…”