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.
Highlights
Helicases are classically defined as motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or DNA-RNA hybrid) using energy derived from ATP hydrolysis
To determine the functions of the Q motif in ChlR1 protein, we changed the glutamine to alanine and transfected the plasmid to HEK 293T cells using PEI
We recently found that ChlR1 helicase has a strong preference for a two-stranded antiparallel G4 substrate (G2’) [32]; we examined the ChlR1-Q23A unwinding activity of this substrate
Summary
Helicases are classically defined as motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or DNA-RNA hybrid) using energy derived from ATP hydrolysis. Helicases play roles in virtually all aspects of nucleic acid metabolism, including DNA replication, repair, recombination, transcription, chromosome segregation, and telomere maintenance [1,2,3]. According to their substrates, helicases are classified as DNA or RNA. Because of their biological importance, helicase defects have been linked to various genetic disorders and cancers [4,5,6]
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