title: The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase.
abstract: 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 sequence motifs (I, Ia, II, III, IV, V, and VI). The Q motif, 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 helicase motifs, the role of the Q motif is less acknowledged. Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw Breakage Syndrome, which is characterized by cellular defects in genome maintenance. 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. ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability. The mutant showed impaired ATPase activity but normal ATP binding. A thermal 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 Q motif is involved in protein dimerization. Taken together, our results suggest that the Q motif is involved in DNA binding but not ATP binding in ChlR1 helicase.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.
authors.forename: Hao
authors.initials: H
authors.lastname: Ding
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.
authors.forename: Manhong
authors.initials: M
authors.lastname: Guo
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.
authors.forename: Venkatasubramanian
authors.initials: V
authors.lastname: Vidhyasagar
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.
authors.forename: Tanu
authors.initials: T
authors.lastname: Talwar
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding
chemical_list: D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C101830:DDX11 protein, human; D053487:DEAD-box RNA Helicases