Ku complex interacts with and stimulates the Werner protein
Abstract
Werner syndrome (WS) is the hallmark premature aging disorder in which affected humans appear older than their chronological age. The protein WRNp, defective in WS, has helicase function, DNA-dependent ATPase, and exonuclease activity. Although WRNp functions in nucleic acid metabolism, there is little or no information about the pathways or protein interactions in which it participates. Here we identify Ku70 and Ku86 as proteins that interact with WRNp. Although Ku proteins had no effect on ATPase or helicase activity, they strongly stimulated specific exonuclease activity. These results suggest that WRNp and the Ku complex participate in a common DNA metabolic pathway.
Werner syndrome (WS) is a human premature aging disorder characterized by the early display of many of the signs and symptoms that are associated with the normal aging process. Cells from WS patients also show a number of phenotypic changes typical of chronologically older normal cells including increased chromosomal abnormalities and rapid onset of cellular senescence (Martin 1997). WS cells are not generally sensitive to DNA-damaging agents but are hypersensitive to the carcinogen 4 nitroquinoline (4NQO) and topoisomerase I-specific inhibitor, campthothecin (Lebel and Leder 1998). The WS gene WRN (Yu et al. 1996) is a member of the RecQ family that includes the RecQ4 and BLM genes, defects that are responsible for the premature aging and cancer prone phenotypes of Rothmund-Thomson (Kitao et al. 1999) and Bloom syndrome (Ellis 1997), respectively. These syndromes are characterized by a high degree of genomic instability, including chromosomal breaks, multiple large deletions, and translocations (Moser et al. 1999). WRNp has helicase activity and an associated DNA-dependent ATPase activity (Gray et al. 1997; Brosh et al. 1999). WRNp also has an exonuclease function located in its amino-terminal region. Two laboratories have identified the directionality of the exonuclease to be 3′ → 5′ (Huang et al. 1998; Kamath-Loeb et al. 1998; Shen et al. 1998), whereas one laboratory reported a 5′ → 3′ directionality (Suzuki et al. 1999).
The enzymatic functions of WRNp indicate a role in nucleic acid metabolism, but information about the pathways in which it participates is lacking. In this study we used a chromatographic procedure to isolate proteins that bind to WRNp. This approach identified Ku70 and Ku86. This physical interaction between the Ku86/70 complex was verified by immunoprecipitation from cell extracts. Moreover, the Ku86/70 complex functionally interacts with WRNp, strongly stimulating the 3′ → 5′ exonuclease activity of WRNp.
Acknowledgments
We thank J. Campisi for the WRN exonuclease mutant baculovirus. D.R. was a recipient of a grant, RPG GMC-98562 from the American Cancer Society.
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Footnotes
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