Search

Search Results

1 results found. Results per page: [ 20 ][ 40 ][ 60 ][ 80 ][ 100 ][ 200 ][ 300 ]

Sort by: [ Publication Date ][ Score ]

Year of publication: [ 2025 ][ 2024 ][ 2023 ][ 2022 ][ 2021 ][ 2020 ][ 2019 ][ 2018 ][ 2017 ][ 2016 ][ 2015 ][ 2014 ][ 2013 ][ 2012 ][ 2011 ][ 2010 ][ 2009 ][ Any ]

Direction: [ Desc ][ Asc ]

• Research Paper Volume 4, Issue 6 pp 417-429

  DNA binding residues in the RQC domain of Werner protein are critical for its catalytic activities

  Relevance score: 12.889217

  Takashi Tadokoro, Tomasz Kulikowicz, Lale Dawut, Deborah L. Croteau, Vilhelm A. Bohr

  Keywords: RecQ helicase, WRN, RQC domain, WH-motif, DNA unwinding, exonuclease

  Published in Aging on June 13, 2012

  Show abstract
  Hide abstract

  Werner protein (WRN), member of the RecQ helicase family, is a helicase and exonuclease, and participates in multiple DNA metabolic processes including DNA replication, recombination and DNA repair. Mutations in the WRN gene cause Werner syndrome, associated with premature aging, genome instability and cancer predisposition. The RecQ C-terminal (RQC) domain of WRN, containing α2-α3 loop and β-wing motifs, is important for DNA binding and for many protein interactions. To better understand the critical functions of this domain, we generated recombinant WRN proteins (using a novel purification scheme) with mutations in Arg-993 within the α2-α3 loop of the RQC domain and in Phe-1037 of the μ-wing motif. We then studied the catalytic activities and DNA binding of these mutant proteins as well as some important functional protein interactions. The mutant proteins were defective in DNA binding and helicase activity, and interestingly, they had deficient exonuclease activity and strand annealing function. The RQC domain of WRN has not previously been implicated in exonuclease or annealing activities. The mutant proteins could not stimulate NEIL1 incision activity as did the wild type. Thus, the Arg-993 and Phe-1037 in the RQC domain play essential roles in catalytic activity, and in functional interactions mediated by WRN.

  

  (A) Domain structure of WRN. Functional domains are indicated below the structure. (B) Amino acid sequence alignment of RQC domain of RecQ helicases. Human WRN RQC (WRN), human BLM RQC (BLM), human RECQL1 RQC (RECQ1) and E. coli RecQ RQC (eRECQ) are shown. Secondary structures of WRN RQC domain are represented below the sequences. ▼ indicates DNA-contacting amino acid residues in WRN RQC-DNA complex (PDB ID: 3AAF). Mutated sites indicated with broken line box. (C) Structure of WRN RQC-DNA complex (PDB ID: 3AAF). The figure generated using PyMOL (DeLano, http://pymol.sourceforge.net/).

  
  
  

  

  (A) The map of 6xHis-WRN-FLAG/pFastBac1-InteinCBDAla construct, total size 9857 bp. (B) Improved WRN purification scheme. (C) SDS-PAGE showing steps of WRN purification. Lane 1, molecular marker; lane 2, whole cell extract; lane 3, pooled fractions from HisTrap column; lane 4, pooled fractions from chitin column. (D) SDS-PAGE of purified WRN variants. Lane 1, molecular marker; lane 2, WRN wild type; lane 3, WRN R993A; lane 4, WRN F1037A; lane 5, WRN R993A/F1037A. (E) Heat denaturation curves of WRN variants. 2 μg of WRN or WRN mutant was incubated with serial dilutions of SYPRO Orange. The curves were obtained by monitoring the change of fluorescence intensity given by qPCR equipment. Observed data was normalized as described in Experimental Procedures.

  
  
  

  

  (A) DNA unwinding activity of WRN variants on forked duplex substrate. 1, 2, 5 nM WRN wild type (lane 2 to 4) or WRN RQC variants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with 0.5 nM DNA substrate at 37 °C for 30 min. Reaction products were separated on 8% polyacrylamide gel. D indicates heat-denatured substrate control. (B) Quantitative analysis of (A). Experiments were repeated at least three times, error bars represent ± SD. (C) Electro mobility shift assay (EMSA) using forked duplex. 1, 2, 5 nM WRN wild type (lane 2 to 4) or WRN variants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with 0.5 nM forked duplex substrate on ice for 30 min, then products were separated on 4 % polyacrylamide gel. (D) ATPase hydrolysis is disrupted by mutations. Representative polyethyleneimine thin-layer chromatography plate of ATP hydrolysis by wild type WRN or mutant proteins in the presence or in the absence of dsDNA is shown.

  
  
  

  

  DNA unwinding activity on (A) Holliday junction (HJ) like substrate, (B) G-quadruplex (G4) substrate, (C) D-loop substrate and (D) bubble substrate. 1, 2, 5 nM WRN wild type (lane 2 to 4) or WRN mutants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with either HJ, G4 or bubble substrate. 5, 10, 20 nM WRN wild type or WRN mutants were incubated with D-loop substrate. Reactions were performed as described in Experimental Procedures. Δ indicates heat-denatured substrate control. (E), (F), (G), (H) Quantitative analysis corresponding to (A), (B), (C), (D), respectively. Experiments were repeated at least three times, error bars represent ± SD.

  
  
  

  

  (A) Exonuclease activity of WRN mutants was severely reduced. 5, 10, 20 nM WRN wild type (lane 2 to 4) or WRN variants (lane 5 to 7, R993A; lane 8 to 10, F1037A; lane 11 to 13, R993A/F1037A) were incubated with 0.5 nM 5' overhang duplex substrate. Products were separated on 14 % denaturing polyacrylamide gel. (B) Ku70/80 heterodimeric protein is not able to stimulate exonuclease activity of WRN mutants. 10 nM WRN wild type (lane 4 to 6) or WRN mutants (lane 7 to 9, R993A; lane 10 to 12, F1037A; lane 13 to 15, R993A/F1037A) were incubated with 5, 10, 20 nM Ku heterodimer and substrate. (C) WRN variants exhibit significantly lower strand annealing activity than the WRN wild type protein. 1, 2, 5 nM WRN wild type or WRN RQC variants were incubated with 0.5 nM DNA substrate at 37 °C for 15 min.

  
  
  

  

  (A) WRN mutants are able to stimulate DNA unwinding activity of wild type WRN by forming hetero oligomeric structure. 2 nM of either native or heat-denatured WRN wild type (2d, heat denatured form) was mixed with 4 nM of either native or heat-denatured WRN mutant (4d, heat denatured form), and incubated with 0.5 nM forked duplex substrate at 37 °C for 20 min. Reaction products were separated on 8% polyacrylamide gel. Δ indicates heat-denatured substrate control. (B) Quantitative analysis of (A). Experiments were repeated at least three times, error bars represent ± SD.

  
  
  

  

  (A) NEIL1 incision activity in the presence of WRN mutants. 1 nM of NEIL1 was incubated with 0, 1, 2 or 4 nM of WRN wild type (lane 3 to 5) or WRN variants (lane 6 to 8, R993A; lane 9 to 11, F1037A; lane 12 to 14, R993A/F1037A) in the presence of 0.5 nM of DNA substrate at 37 °C for 10 min. Reaction products were separated on 10 % denaturing polyacrylamide gel. (B) Quantitative analysis of (A). Experiments were repeated at least three times, error bars represent ± SD.