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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

ChlR1-Q23A fails to unwind DNA triple helixes.(A-B) Helicase reactions (20 μL) were performed by incubating the indicated ChlR1-WT (A) or ChlR1-Q23A (B) concentrations with 0.5 nM 5’ tail plasmid-triplex substrate at 37°C for 20 min under standard helicase assay conditions as described in “Materials and methods”. Triangle indicates heat-denatured DNA substrate control.
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pone.0140755.g005: ChlR1-Q23A fails to unwind DNA triple helixes.(A-B) Helicase reactions (20 μL) were performed by incubating the indicated ChlR1-WT (A) or ChlR1-Q23A (B) concentrations with 0.5 nM 5’ tail plasmid-triplex substrate at 37°C for 20 min under standard helicase assay conditions as described in “Materials and methods”. Triangle indicates heat-denatured DNA substrate control.

Mentions: Triplex displacement experiments have been utilized to monitor the translocase activity of helicases, including AddAB [43] and FANCM [44]. In this assay, a triple helix is formed when a third strand forms Hoogsteen base pairs with duplex DNA. If a translocase proceeds through the triplex, it will displace the third strand. We found that wild-type ChlR1, but not its Q mutant Q23A, displayed triple-helix displacement activity (Fig 5A and 5B). Under the same reaction conditions, ChlR1-WT was also able to unwind a short triplex structure (named flush triplex), which was constructed by annealing the same pyrimidine motif third strand (TC30) to a 30 bp duplex fragment; however, the mutant ChlR1-Q23A failed in this regard (S3 Fig). Together, these results suggest that ChlR1 can dissociate DNA triplexes; however, the Q23A mutant has no translocase activity.


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)

ChlR1-Q23A fails to unwind DNA triple helixes.(A-B) Helicase reactions (20 μL) were performed by incubating the indicated ChlR1-WT (A) or ChlR1-Q23A (B) concentrations with 0.5 nM 5’ tail plasmid-triplex substrate at 37°C for 20 min under standard helicase assay conditions as described in “Materials and methods”. Triangle indicates heat-denatured DNA substrate control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140755.g005: ChlR1-Q23A fails to unwind DNA triple helixes.(A-B) Helicase reactions (20 μL) were performed by incubating the indicated ChlR1-WT (A) or ChlR1-Q23A (B) concentrations with 0.5 nM 5’ tail plasmid-triplex substrate at 37°C for 20 min under standard helicase assay conditions as described in “Materials and methods”. Triangle indicates heat-denatured DNA substrate control.
Mentions: Triplex displacement experiments have been utilized to monitor the translocase activity of helicases, including AddAB [43] and FANCM [44]. In this assay, a triple helix is formed when a third strand forms Hoogsteen base pairs with duplex DNA. If a translocase proceeds through the triplex, it will displace the third strand. We found that wild-type ChlR1, but not its Q mutant Q23A, displayed triple-helix displacement activity (Fig 5A and 5B). Under the same reaction conditions, ChlR1-WT was also able to unwind a short triplex structure (named flush triplex), which was constructed by annealing the same pyrimidine motif third strand (TC30) to a 30 bp duplex fragment; however, the mutant ChlR1-Q23A failed in this regard (S3 Fig). Together, these results suggest that ChlR1 can dissociate DNA triplexes; however, the Q23A mutant has no translocase activity.

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