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

Ding H, Guo M, Vidhyasagar V, Talwar T, Wu Y - PLoS ONE (2015)

Bottom Line: ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.

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.

No MeSH data available.


Related in: MedlinePlus

DNA binding analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.The indicated concentrations of ChlR1 proteins were incubated with 0.5 nM forked duplex DNA substrate (A-C) or OX-1 G2’ DNA substrate (D-E) at room temperature for 30 min under standard EMSA conditions as described in “Materials and methods”. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. (F) Variation of fluorescence anisotropy as a function of ChlR1 protein concentration. (G) A representative image of filter dot blot assays of ChlR1 proteins binding forked duplex DNA. (H) Quantitative analyses of DNA bound to ChlR1 proteins in panel G. Data represent the mean of at least three independent experiments with SD indicated by error bars.
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pone.0140755.g003: DNA binding analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.The indicated concentrations of ChlR1 proteins were incubated with 0.5 nM forked duplex DNA substrate (A-C) or OX-1 G2’ DNA substrate (D-E) at room temperature for 30 min under standard EMSA conditions as described in “Materials and methods”. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. (F) Variation of fluorescence anisotropy as a function of ChlR1 protein concentration. (G) A representative image of filter dot blot assays of ChlR1 proteins binding forked duplex DNA. (H) Quantitative analyses of DNA bound to ChlR1 proteins in panel G. Data represent the mean of at least three independent experiments with SD indicated by error bars.

Mentions: The ability of the ChlR1-Q23A mutant protein to abolish helicase activity might reflect impaired DNA binding activity. To test this assumption, we performed electrophoretic mobility shift assays with ChlR1-WT and ChlR1-Q23A using radiolabeled DNA substrates that were used in the helicase assay. Results demonstrated that ChlR1-WT bound the DNA molecules in a protein concentration-dependent manner while the binding ability of the ChlR1-Q23A protein was abolished (Fig 3A and 3B). When the protein concentration was increased by up to eight times (96 nM), ChlR1-Q23A could slightly bind the substrate (Fig 3C). Compared with forked duplex DNA, ChlR1-WT had a lower binding ability to the G2’ substrate; however, ChlR1-Q23A completely lost its binding ability to this G2’ structure (Fig 3D and 3E), even at an increased protein concentration (240 nM, data not shown). Thus, we concluded that the Q motif is essential for ChlR1 protein’s DNA binding ability.


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

Ding H, Guo M, Vidhyasagar V, Talwar T, Wu Y - PLoS ONE (2015)

DNA binding analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.The indicated concentrations of ChlR1 proteins were incubated with 0.5 nM forked duplex DNA substrate (A-C) or OX-1 G2’ DNA substrate (D-E) at room temperature for 30 min under standard EMSA conditions as described in “Materials and methods”. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. (F) Variation of fluorescence anisotropy as a function of ChlR1 protein concentration. (G) A representative image of filter dot blot assays of ChlR1 proteins binding forked duplex DNA. (H) Quantitative analyses of DNA bound to ChlR1 proteins in panel G. Data represent the mean of at least three independent experiments with SD indicated by error bars.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608764&req=5

pone.0140755.g003: DNA binding analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.The indicated concentrations of ChlR1 proteins were incubated with 0.5 nM forked duplex DNA substrate (A-C) or OX-1 G2’ DNA substrate (D-E) at room temperature for 30 min under standard EMSA conditions as described in “Materials and methods”. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. (F) Variation of fluorescence anisotropy as a function of ChlR1 protein concentration. (G) A representative image of filter dot blot assays of ChlR1 proteins binding forked duplex DNA. (H) Quantitative analyses of DNA bound to ChlR1 proteins in panel G. Data represent the mean of at least three independent experiments with SD indicated by error bars.
Mentions: The ability of the ChlR1-Q23A mutant protein to abolish helicase activity might reflect impaired DNA binding activity. To test this assumption, we performed electrophoretic mobility shift assays with ChlR1-WT and ChlR1-Q23A using radiolabeled DNA substrates that were used in the helicase assay. Results demonstrated that ChlR1-WT bound the DNA molecules in a protein concentration-dependent manner while the binding ability of the ChlR1-Q23A protein was abolished (Fig 3A and 3B). When the protein concentration was increased by up to eight times (96 nM), ChlR1-Q23A could slightly bind the substrate (Fig 3C). Compared with forked duplex DNA, ChlR1-WT had a lower binding ability to the G2’ substrate; however, ChlR1-Q23A completely lost its binding ability to this G2’ structure (Fig 3D and 3E), even at an increased protein concentration (240 nM, data not shown). Thus, we concluded that the Q motif is essential for ChlR1 protein’s DNA binding ability.

Bottom Line: ChlR1-Q23A mutant abolished the helicase activity of ChlR1 and displayed reduced DNA binding ability.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Saskatchewan, Health Sciences Building, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada.

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.

No MeSH data available.


Related in: MedlinePlus