Ku complex interacts with and stimulates the Werner protein - PubMed (original) (raw)

. 2000 Apr 15;14(8):907-12.

Affiliations

PMID: 10783163
PMCID: PMC316545

Ku complex interacts with and stimulates the Werner protein

M P Cooper et al. Genes Dev. 2000.

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.

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Figures

Figure 1

Purification of the carboxyl terminus of Werner protein. (A) Schematic of C-WRNp fragment. The amino terminus contains the 3 conserved exonuclease motifs. The central region contains the conserved 7 motif helicase domain. (B) SDS-PAGE with Coomassie staining of C-WRNp.

Figure 1

Figure 2

Specific proteins bound to C-WRNp. C-WRNp binds to full-length WRNp (∼160 kD). Western blot with anti-WRNp antibody. (Lane 1) Nuclear extract, positive control; (lane 2) β-galactosidase column eluate; (lane 3) C-WRNp column eluate.

Figure 3

Ku proteins interact with C-WRNp. (A) Two proteins tightly associated with C-WRNp were identified by Coomassie staining of samples analyzed by SDS-PAGE. (Lane 1) Marker; (lane 2) no protein column; (lane 3) β-galactosidase column; (lane 4) C-WRNp column. (Arrows) Proteins that associated with C-WRNp specifically. (B) Western analysis using antibodies against Ku86 and Ku70 identify the two specific proteins as the Ku proteins. Antibodies against Ku86 and Ku70 were obtained from Oncogene Research Products (Cambridge, MA) and used to sequentially probe the membrane.

Figure 4

WRNp and Ku complex coimmunoprecipitate. (A) Polyclonal antibody raised against the amino terminus of WRN (amino acids 1–600, designated N-WRN) was raised in mouse. The blot was sequentially probed with the Ku86 and Ku70 antibodies. (Lane 1) Anti-WRN mouse polyclonal antibody immunoprecipitate; (lane 2) anti-Ku70 mouse monoclonal antibody immunoprecipitate; (lane 3) anti-Ku86 mouse monoclonal antibody immunoprecipitate; (lane 4) anit-Ku70/86 goat polyclonal antibody immunoprecipitate; (lane 5) goat immunoglobin precipitate; (lane 6) mouse immunoglobin precipitate; (lane 7) blank; (lane 8) HeLa nuclear extract, unprecipitated, to mark the positions of Ku86 and Ku70. (B) Immunoprecipitation of WRNp by antibodies specific for N-WRN, Ku70, Ku86, and the Ku86/70 heterodimer. The insert labeled WRN (lane 1) shows the anti-N-WRN antibody immunoprecipitate probed with polycolonal antibodies against the carboxyl terminus of WRN (prepared similarly to the anti-N WRN antibody), demonstrating that the anti-N-WRN antibody is specific for full-length WRN. (Lane 2) Anti-Ku70 mouse mAb immunoprecipitate; (lane 3) anti-Ku86 mouse mAb immunoprecipitate; (lane 4) anti-Ku86/70 goat polyclonal antibody immunoprecipitate; (lane 5) mouse immunoglobulin immunoprecipitate; (lane 6) HeLa nuclear extract, unprecipitated, to mark the position of WRNp.