RESEARCH ARTICLE The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding of structured DNA or RNA and chromatin remodeling. The conversion of energy derived from ATP hydrolysis into unwinding and remodeling is coordinated by seven sequencemotifs (I, Ia, II, III, IV, V, and VI). The Qmotif, consisting of nine amino acids (GFXXPXPIQ) with an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Compared to the seven well-recognized conserved helicasemotifs, the role of the Qmotif is less acknowledged. Mutations in the humanChlR1 (DDX11) gene are associated with a unique genetic disorder known asWarsaw Breakage Syndrome, which is characterized by cellular defects in genomemaintenance. To examine the roles of the Qmotif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Qmotif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. ChlR1-Q23Amutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A ther-mal shift assay revealed that ChlR1-Q23A has a melting point value similar to ChlR1-WT. Partial proteolysis mapping demonstrated that ChlR1-WT and Q23A have a similar globular structure, although some subtle conformational differences in these two proteins are evident. Finally, we found ChlR1 exists and functions as a monomer in solution, which is different from FANCJ, in which the Qmotif is involved in protein dimerization. Taken together, our results suggest that the Qmotif is involved in DNA binding but not ATP binding in ChlR1 helicase