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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

Purification and identification of ChlR1 proteins.(A) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. (B-C) Western-blot analysis of the purified proteins with antibodies ChlR1 (B) and FLAG (C).
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pone.0140755.g001: Purification and identification of ChlR1 proteins.(A) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. (B-C) Western-blot analysis of the purified proteins with antibodies ChlR1 (B) and FLAG (C).

Mentions: Close inspection of human ChlR1 and its orthologs across species revealed a conserved glutamine (Q motif) residue upstream of motif I (S1 Fig). To determine the functions of the Q motif in ChlR1 protein, we changed the glutamine to alanine (designated ChlR1-Q23A) and transfected the plasmid to HEK 293T cells using PEI. The wild-type and mutant ChlR1 proteins were purified to near homogeneity as judged by their appearance as single bands after electrophoresis on Coomassie blue-stained SDS-polyacrylamide gel (Fig 1A). The protein identity was confirmed by Western blot using an anti-ChlR1 antibody and anti-FLAG antibody, respectively (Fig 1B and 1C).


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)

Purification and identification of ChlR1 proteins.(A) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. (B-C) Western-blot analysis of the purified proteins with antibodies ChlR1 (B) and FLAG (C).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140755.g001: Purification and identification of ChlR1 proteins.(A) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. (B-C) Western-blot analysis of the purified proteins with antibodies ChlR1 (B) and FLAG (C).
Mentions: Close inspection of human ChlR1 and its orthologs across species revealed a conserved glutamine (Q motif) residue upstream of motif I (S1 Fig). To determine the functions of the Q motif in ChlR1 protein, we changed the glutamine to alanine (designated ChlR1-Q23A) and transfected the plasmid to HEK 293T cells using PEI. The wild-type and mutant ChlR1 proteins were purified to near homogeneity as judged by their appearance as single bands after electrophoresis on Coomassie blue-stained SDS-polyacrylamide gel (Fig 1A). The protein identity was confirmed by Western blot using an anti-ChlR1 antibody and anti-FLAG antibody, respectively (Fig 1B and 1C).

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