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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

Thermal stability assays and partial proteolysis mapping of ChlR1 proteins.(A) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. (B) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.
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pone.0140755.g006: Thermal stability assays and partial proteolysis mapping of ChlR1 proteins.(A) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. (B) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.

Mentions: To address whether ChlR1-Q23A protein has a similar structure to the wild type, we attempted to use circular dichroism to determine the secondary structure and folding properties of proteins but failed because circular dichroism analysis requires a significant amount of concentrated protein and specific buffer. In contrast, thermal shift assays are widely used to measure the thermal stability of target proteins [45–47], which indirectly reflect protein folding. Thus, we performed a thermal shift assay for the purified recombinant ChlR1 proteins. In the temperature range from 25 to 60°C, we found that ChlR1-Q23A protein exhibited a similar transition curve to the wild-type protein (Fig 6A). Melting curve analysis with a derivative reporter showed that the midpoint (Tm) for both ChlR1-WT and ChlR1-Q23A was 43.9°C, with a ΔTm of ~0°C, suggesting that ChlR1-Q23A protein’s globular structure is similar to that of ChlR1-WT.


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)

Thermal stability assays and partial proteolysis mapping of ChlR1 proteins.(A) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. (B) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140755.g006: Thermal stability assays and partial proteolysis mapping of ChlR1 proteins.(A) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. (B) Representative image of partial proteolysis mapping of ChlR1 proteins. Purified ChlR1 proteins (WT and Q23A) were digested with increasing trypsin concentration, and protein fragments were separated on SDS-PAGE followed by Western blot analysis using an anti-FLAG antibody.
Mentions: To address whether ChlR1-Q23A protein has a similar structure to the wild type, we attempted to use circular dichroism to determine the secondary structure and folding properties of proteins but failed because circular dichroism analysis requires a significant amount of concentrated protein and specific buffer. In contrast, thermal shift assays are widely used to measure the thermal stability of target proteins [45–47], which indirectly reflect protein folding. Thus, we performed a thermal shift assay for the purified recombinant ChlR1 proteins. In the temperature range from 25 to 60°C, we found that ChlR1-Q23A protein exhibited a similar transition curve to the wild-type protein (Fig 6A). Melting curve analysis with a derivative reporter showed that the midpoint (Tm) for both ChlR1-WT and ChlR1-Q23A was 43.9°C, with a ΔTm of ~0°C, suggesting that ChlR1-Q23A protein’s globular structure is similar to that of ChlR1-WT.

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