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Mutations in The RQC Domain of Human WRN Helicase Compromise It's Ability To Unwind G‐Quadruplex DNA
Author(s) -
Ketkar Amit,
Voehler Markus,
Mukiza Tresor,
Eoff Robert Lawton
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.576.3
Subject(s) - helicase , dna replication , dna , werner syndrome , recq helicase , biology , microbiology and biotechnology , holliday junction , replication protein a , genetics , dna repair , dna binding protein , dna damage , genome instability , gene , rna , transcription factor
G‐quadruplex (G4) DNA is a specialized DNA structure that is now well established to occur in vivo . G4‐DNA structures present a significant block for replication, and must be overcome to complete the process efficiently. G4 motifs have been shown to occur at functional regions of prokaryotic and eukaryotic genomes, like promoters of oncogenes, replication origins and telomeres. G4 sequences are also enriched at chromosomal breakpoints in multiple types of cancer. The Werner's syndrome protein (WRN) is a RecQ‐family helicase that has been implicated in efficiently unwinding G4‐DNA structures during replication in humans. The aim of our study was to identify the residues of WRN that were involved in this role. Using a c‐Myc G4‐DNA model sequence and recombinant WRN, we determined that the RecQ‐C‐terminal (RQC) domain of WRN imparted a 2‐fold preference for binding to G4‐DNA relative to non‐G4 DNA substrates. NMR spectroscopic studies were performed using 15 N‐labeled RQC‐domain in DNA titration experiments where both non‐structured and G4‐DNA substrates were used to identify residues that are involved specifically in interacting with G4‐DNA. Residues that showed the largest G4‐specific changes in NMR signal were chosen for further validation. Point mutations were made in these RQC‐domain residues in the WRN construct 500–1092, to generate single‐mutant proteins WRN T1024G , WRN L1063G , WRN T1086G , and a double‐mutant WRN T1024G/T1086G . The mutant proteins were studied in detail for their ATPase activity, ability to unwind G4‐DNA and their relative affinities towards non‐structured or G4‐DNA substrates. ATPase activity of all mutant proteins was reduced compared to wild‐type. The effect was moderate when nonG4‐DNA was used, and most pronounced for the double mutant when G4‐ssDNA was used to stimulate ATPase activity (activity was only 40% of wild‐type). The helicase/DNA‐unwinding activity of all mutant proteins was relatively unaffected on a non‐G4‐DNA substrate. However, for the WRN T1024G mutant, this activity was reduced by ~50% on a G4‐DNA substrate, while for the double mutant, it was drastically reduced (by 90%) on G4‐DNA as compared to wild‐type. DNA‐binding affinity of all mutant proteins except WRN T1024G/T1086G was relatively unaffected for both control and G4‐DNA substrates. Binding preference towards G4‐DNA was reduced only moderately for the single mutants. In the case of the double mutant, the preference for G4‐DNA was reduced from ~2‐fold (in wild‐type) to ~1.1‐fold. Our work has, thus, for the first time, helped identify the residues of WRN that are involved specifically in its interaction with G4‐DNA, and has laid the groundwork for future studies on properties of RecQ‐helicases, which enable several of them to unwind G‐quadruplex DNA. Support or Funding Information Grants GM084460, CA183895