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The Q Motif is Involved in DNA Binding in ChlR1 (DDX11) Helicase

Helicases are molecular motors that couple the energy of nucleoside triphosphate (NTP) hydrolysis to the unwinding and remodeling of structured DNA or RNA. The conversion of energy derived from NTP hydrolysis into unwinding of double‐stranded nucleic acids is coordinated by seven sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, consisting of an invariant glutamine (Q) residue, has been identified in some, but not all helicases. Previous studies have suggested that the Q motif is involved in ATP binding. Mutations in the human ChlR1 (DDX11) gene are associated with Warsaw Breakage Syndrome characterized by cellular defects in genome maintenance. ChlR1 is known to play essential roles to preserve genomic stability, particularly in sister chromatid cohesion; however, its molecular mechanism remains unknown. To examine the roles of the Q motif in ChlR1 helicase, we performed site directed mutagenesis of glutamine to alanine at residue 23 in the Q motif of ChlR1. ChlR1 recombinant protein was overexpressed and purified from HEK293T cells. The ChlR1‐Q23A mutant abolished helicase activity of ChlR1, and further DNA binding assays suggested the Q motif affected the DNA binding ability of ChlR1. ATP hydrolysis assays revealed that the ChlR1‐Q23A had impaired ATP hydrolysis activity. However, further ATP binding assays showed that the Q motif is not essential for ATP binding. The Q motif in FANCJ helicase, a ChlR1 sequence homology protein, regulates FANCJ's dimerization, while our size exclusion chromatography indicated that ChlR1 protein functions as monomer. Taken together, our results suggest that the Q motif in ChlR1 helicase is involved in DNA binding but not ATP binding.