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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

Helicase analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.Helicase reactions were performed by incubating with indicated protein concentration and 0.5 nM duplex DNA substrate (A-B) or OX-1 G2’ DNA substrate (C-D) at 37°C for 20 min. The triangle indicates heat denatured DNA substrate control.
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pone.0140755.g002: Helicase analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.Helicase reactions were performed by incubating with indicated protein concentration and 0.5 nM duplex DNA substrate (A-B) or OX-1 G2’ DNA substrate (C-D) at 37°C for 20 min. The triangle indicates heat denatured DNA substrate control.

Mentions: Using a forked duplex DNA substrate that we know ChlR1-WT can efficiently unwind [32,35], we examined the helicase activity of ChlR1-Q23A. The results revealed that changing the invariant glutamine to alanine of the Q motif abolished ChlR1’s unwinding activity on forked duplex DNA (Fig 2A and 2B). Approximately 100% of the forked duplex was unwound at the highest concentration of 1.2 nM ChlR1-WT; however, ChlR1-Q23A failed to unwind the duplex substrate at the same concentration. We recently found that ChlR1 helicase has a strong preference for a two-stranded antiparallel G4 substrate (G2’) [32]; thus, we also examined the ChlR1-Q23A unwinding activity of this substrate. ChlR1-WT was able to unwind more than 60% of the substrate at its highest concentration (24 nM); however, ChlR1-Q23A failed to unwind this substrate at the same concentration (Fig 2C and 2D). We also increased the incubation time (60 min), ATP concentration (10 mM), or protein concentration (to 12 nM for forked duplex DNA and 240 nM for G2’ substrate) but failed to detect any unwinding activity for ChlR1-Q23A (data not shown). Taken together, we concluded that the ChlR1-Q23A mutant abolishes the helicase 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)

Helicase analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.Helicase reactions were performed by incubating with indicated protein concentration and 0.5 nM duplex DNA substrate (A-B) or OX-1 G2’ DNA substrate (C-D) at 37°C for 20 min. The 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.g002: Helicase analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.Helicase reactions were performed by incubating with indicated protein concentration and 0.5 nM duplex DNA substrate (A-B) or OX-1 G2’ DNA substrate (C-D) at 37°C for 20 min. The triangle indicates heat denatured DNA substrate control.
Mentions: Using a forked duplex DNA substrate that we know ChlR1-WT can efficiently unwind [32,35], we examined the helicase activity of ChlR1-Q23A. The results revealed that changing the invariant glutamine to alanine of the Q motif abolished ChlR1’s unwinding activity on forked duplex DNA (Fig 2A and 2B). Approximately 100% of the forked duplex was unwound at the highest concentration of 1.2 nM ChlR1-WT; however, ChlR1-Q23A failed to unwind the duplex substrate at the same concentration. We recently found that ChlR1 helicase has a strong preference for a two-stranded antiparallel G4 substrate (G2’) [32]; thus, we also examined the ChlR1-Q23A unwinding activity of this substrate. ChlR1-WT was able to unwind more than 60% of the substrate at its highest concentration (24 nM); however, ChlR1-Q23A failed to unwind this substrate at the same concentration (Fig 2C and 2D). We also increased the incubation time (60 min), ATP concentration (10 mM), or protein concentration (to 12 nM for forked duplex DNA and 240 nM for G2’ substrate) but failed to detect any unwinding activity for ChlR1-Q23A (data not shown). Taken together, we concluded that the ChlR1-Q23A mutant abolishes the helicase 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