Abstract
The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ encodes a Q motif DEAH box DNA helicase implicated in Fanconi anemia and breast cancer. A Q25A mutation of the invariant glutamine in the Q motif abolished its ability to complement cisplatin or telomestatin sensitivity of a fancj null cell line and exerted a dominant negative effect. Biochemical characterization of the purified recombinant FANCJ-Q25A protein showed that the mutation disabled FANCJ helicase activity and the ability to disrupt protein-DNA interactions. FANCJ-Q25A showed impaired DNA binding and ATPase activity but displayed ATP binding and temperature-induced unfolding transition similar to FANCJ-WT. Size exclusion chromatography and sedimentation velocity analyses revealed that FANCJ-WT existed as molecular weight species corresponding to a monomer and a dimer, and the dimeric form displayed a higher specific activity for ATPase and helicase, as well as greater DNA binding. In contrast, FANCJ-Q25A existed only as a monomer, devoid of helicase activity. Thus, the Q motif is essential for FANCJ enzymatic activity in vitro and DNA repair function in vivo.
Highlights
The conserved Q motif of RNA/DNA helicases has been structurally implicated in nucleotide binding; its biochemical functions are less certain
These results confirm the findings from the size exclusion chromatography analysis, suggesting that FANCJ-WT exists in two populations distinguished by molecular weight and relative catalytic activity, whereas FANCJQ25A is found only in a lower molecular weight form that is devoid of helicase activity
Previous work suggested that the Q motif in many RNA and DNA helicases plays an important role in nucleotide binding; our biochemical studies of the FANCJ-Q25A mutant protein indicate that deletion of the Q motif or replacement of the conserved glutamine with an alanine affected DNA binding in addition to ATP hydrolysis
Summary
The conserved Q motif of RNA/DNA helicases has been structurally implicated in nucleotide binding; its biochemical functions are less certain. The Q motif, conserved in a number of RNA and DNA helicases, is proposed to be important for ATP binding based on structural data, but its precise biochemical functions are less certain. FANCJ is a DNA-stimulated ATPase, and mutation of the invariant lysine residue in the conserved motif I (Walker A box) in the helicase core domain of FANCJ abolishes its ATPase activity and DNA unwinding of simple partial duplex DNA substrates [27, 28]. Replacement of the invariant glutamine in the Q motif of FANCJ with an alanine impaired ATP hydrolysis, abolished FANCJ helicase activity, and eliminated its ability to catalytically strip protein bound to DNA. The biochemical and genetic analyses demonstrate that the Q motif in FANCJ is essential for its catalytic activity and DNA repair function in vivo
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