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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

ATP hydrolysis and ATP binding assays of ChlR1 proteins.(A) A representative image of ChlR1 ATP hydrolysis detected by TLC. (B) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. (C) ATP binding by wild-type ChlR1 and mutant protein. α32P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. (D) A representative image of filter dot blot assays of ChlR1 proteins binding α32P-ATP. (E) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.
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pone.0140755.g004: ATP hydrolysis and ATP binding assays of ChlR1 proteins.(A) A representative image of ChlR1 ATP hydrolysis detected by TLC. (B) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. (C) ATP binding by wild-type ChlR1 and mutant protein. α32P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. (D) A representative image of filter dot blot assays of ChlR1 proteins binding α32P-ATP. (E) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.

Mentions: Most helicases have DNA- or RNA-dependent ATP hydrolysis activity. Thus, we examined the DNA-dependent ATPase activity of ChlR1-Q23A and compared it with ChlR1-WT. Using covalently closed M13 single strand DNA as the effector molecule and ATP ranging from 31 to 4000 μM, we determined the Km value of ATP hydrolysis for ChlR1-WT is 415.7 ± 58.4 μM. Because of the very low ATPase activity, the Km for ChlR1-Q23A could not be determined (Fig 4A). Using an ATP concentration (8.5 mM) that was ∼20-fold greater than the Km for ChlR1-WT, we performed ATPase assays for ChlR1-WT and ChlR1-Q23A and determined the kcat values for these two proteins were 1073.3 ± 46.7 and 110.7 ± 9.3 s-1, respectively. These results suggest that the ChlR1-Q23A mutation seriously compromises the ability of ChlR1 to hydrolyze ATP.


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)

ATP hydrolysis and ATP binding assays of ChlR1 proteins.(A) A representative image of ChlR1 ATP hydrolysis detected by TLC. (B) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. (C) ATP binding by wild-type ChlR1 and mutant protein. α32P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. (D) A representative image of filter dot blot assays of ChlR1 proteins binding α32P-ATP. (E) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. 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.g004: ATP hydrolysis and ATP binding assays of ChlR1 proteins.(A) A representative image of ChlR1 ATP hydrolysis detected by TLC. (B) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. (C) ATP binding by wild-type ChlR1 and mutant protein. α32P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. (D) A representative image of filter dot blot assays of ChlR1 proteins binding α32P-ATP. (E) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.
Mentions: Most helicases have DNA- or RNA-dependent ATP hydrolysis activity. Thus, we examined the DNA-dependent ATPase activity of ChlR1-Q23A and compared it with ChlR1-WT. Using covalently closed M13 single strand DNA as the effector molecule and ATP ranging from 31 to 4000 μM, we determined the Km value of ATP hydrolysis for ChlR1-WT is 415.7 ± 58.4 μM. Because of the very low ATPase activity, the Km for ChlR1-Q23A could not be determined (Fig 4A). Using an ATP concentration (8.5 mM) that was ∼20-fold greater than the Km for ChlR1-WT, we performed ATPase assays for ChlR1-WT and ChlR1-Q23A and determined the kcat values for these two proteins were 1073.3 ± 46.7 and 110.7 ± 9.3 s-1, respectively. These results suggest that the ChlR1-Q23A mutation seriously compromises the ability of ChlR1 to hydrolyze ATP.

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