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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

Determination of ChlR1 protein oligomerization state.(A) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. (B) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. (C) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. (D) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. (E) The fractions in D were immunoblotted with an anti-FLAG antibody. (F) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.
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pone.0140755.g007: Determination of ChlR1 protein oligomerization state.(A) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. (B) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. (C) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. (D) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. (E) The fractions in D were immunoblotted with an anti-FLAG antibody. (F) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.

Mentions: Certain helicases may self-assemble to form dimers or higher order oligomers, which can influence their catalytic or biological functions [1,7,48]. The Q motif in FANCJ helicase regulates its dimerization in solution [49], but the oligomeric state of ChlR1 has never been determined. To determine whether the Q motif might affect oligomerization of ChlR1, we analyzed the recombinant ChlR1 proteins by size exclusion chromatography. The purified ChlR1-WT protein (Fig 7A) was applied to a Sephacryl S-300 HR column, and a major peak was detected at an elution volume of ~65 mL (Fig 7B). Using protein standards, we generated a calibration curve (Fig 7C) and determined the molecular weight of this peak is ~141.1 kDa, which is close but greater than ChlR1’s expected mass (101.7 kDa); this difference in mass may be due to post-translational modification. We then selected 14 fractions from the peak region for SDS-PAGE (Fig 7D) and Western-blot assays (Fig 7E), and both results demonstrated that the absorption peak was contributed by the ChlR1 protein. Moreover, we selected fractions #4 and #5 for helicase activity analysis, and found that this monomeric form of ChlR1 had unwinding activity (Fig 7F); however, they exhibited lower activity than the same protein before size exclusion chromatography, which may be attributed to the overnight size exclusion chromatography procedure.


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)

Determination of ChlR1 protein oligomerization state.(A) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. (B) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. (C) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. (D) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. (E) The fractions in D were immunoblotted with an anti-FLAG antibody. (F) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140755.g007: Determination of ChlR1 protein oligomerization state.(A) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. (B) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. (C) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. (D) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. (E) The fractions in D were immunoblotted with an anti-FLAG antibody. (F) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.
Mentions: Certain helicases may self-assemble to form dimers or higher order oligomers, which can influence their catalytic or biological functions [1,7,48]. The Q motif in FANCJ helicase regulates its dimerization in solution [49], but the oligomeric state of ChlR1 has never been determined. To determine whether the Q motif might affect oligomerization of ChlR1, we analyzed the recombinant ChlR1 proteins by size exclusion chromatography. The purified ChlR1-WT protein (Fig 7A) was applied to a Sephacryl S-300 HR column, and a major peak was detected at an elution volume of ~65 mL (Fig 7B). Using protein standards, we generated a calibration curve (Fig 7C) and determined the molecular weight of this peak is ~141.1 kDa, which is close but greater than ChlR1’s expected mass (101.7 kDa); this difference in mass may be due to post-translational modification. We then selected 14 fractions from the peak region for SDS-PAGE (Fig 7D) and Western-blot assays (Fig 7E), and both results demonstrated that the absorption peak was contributed by the ChlR1 protein. Moreover, we selected fractions #4 and #5 for helicase activity analysis, and found that this monomeric form of ChlR1 had unwinding activity (Fig 7F); however, they exhibited lower activity than the same protein before size exclusion chromatography, which may be attributed to the overnight size exclusion chromatography procedure.

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