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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

SDS-PAGE analysis of ChlR1 proteins by sucrose gradient fractions.Coomassie blue-stained gels of protein standards (A), and silver staining of ChlR1-WT (B) and ChlR1-Q23A (C) from the sucrose gradient centrifugation. Thirty (30) μL of the sucrose-adjusted fractions (1–28) were loaded per lane. The positions of carbonic anhydrase (29 kDa), BSA (66 kDa), and alcohol dehydrogenase (150 kDa) are indicated at the top. Note that alcohol dehydrogenase is a homotetramer, and is shown in subunits of 37.7 kDa after denaturing.
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pone.0140755.g008: SDS-PAGE analysis of ChlR1 proteins by sucrose gradient fractions.Coomassie blue-stained gels of protein standards (A), and silver staining of ChlR1-WT (B) and ChlR1-Q23A (C) from the sucrose gradient centrifugation. Thirty (30) μL of the sucrose-adjusted fractions (1–28) were loaded per lane. The positions of carbonic anhydrase (29 kDa), BSA (66 kDa), and alcohol dehydrogenase (150 kDa) are indicated at the top. Note that alcohol dehydrogenase is a homotetramer, and is shown in subunits of 37.7 kDa after denaturing.

Mentions: To evaluate if the presence of ATP affects its apparent oligomerization properties, we incubated ChlR1-WT protein with AMP-PNP, and applied it to a Sephacryl S-300 HR column. A major peak was detected at an elution volume of ~65 mL (data not shown), suggesting ChlR1 protein still exists as a monomer after it is bound by ATP. We also performed sucrose density gradient centrifugation to determine the molecular mass of both ChlR1-WT and -Q23A mutant. SDS-PAGE analysis showed that both ChlR1-WT and Q23A migrated between fractions of BSA (66 kDa) and alcohol dehydrogenase (150 kDa) (Fig 8). The molecular mass of the ChlR1-WT was calculated as ~135.4 kDa and ChlR1-Q23A mutant is ~144.2 kDa. Taken together, our results suggest that ChlR1 exists and function as a monomer in solution; this, is different that FANCJ, which can function as both a monomer and dimer [49].


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)

SDS-PAGE analysis of ChlR1 proteins by sucrose gradient fractions.Coomassie blue-stained gels of protein standards (A), and silver staining of ChlR1-WT (B) and ChlR1-Q23A (C) from the sucrose gradient centrifugation. Thirty (30) μL of the sucrose-adjusted fractions (1–28) were loaded per lane. The positions of carbonic anhydrase (29 kDa), BSA (66 kDa), and alcohol dehydrogenase (150 kDa) are indicated at the top. Note that alcohol dehydrogenase is a homotetramer, and is shown in subunits of 37.7 kDa after denaturing.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140755.g008: SDS-PAGE analysis of ChlR1 proteins by sucrose gradient fractions.Coomassie blue-stained gels of protein standards (A), and silver staining of ChlR1-WT (B) and ChlR1-Q23A (C) from the sucrose gradient centrifugation. Thirty (30) μL of the sucrose-adjusted fractions (1–28) were loaded per lane. The positions of carbonic anhydrase (29 kDa), BSA (66 kDa), and alcohol dehydrogenase (150 kDa) are indicated at the top. Note that alcohol dehydrogenase is a homotetramer, and is shown in subunits of 37.7 kDa after denaturing.
Mentions: To evaluate if the presence of ATP affects its apparent oligomerization properties, we incubated ChlR1-WT protein with AMP-PNP, and applied it to a Sephacryl S-300 HR column. A major peak was detected at an elution volume of ~65 mL (data not shown), suggesting ChlR1 protein still exists as a monomer after it is bound by ATP. We also performed sucrose density gradient centrifugation to determine the molecular mass of both ChlR1-WT and -Q23A mutant. SDS-PAGE analysis showed that both ChlR1-WT and Q23A migrated between fractions of BSA (66 kDa) and alcohol dehydrogenase (150 kDa) (Fig 8). The molecular mass of the ChlR1-WT was calculated as ~135.4 kDa and ChlR1-Q23A mutant is ~144.2 kDa. Taken together, our results suggest that ChlR1 exists and function as a monomer in solution; this, is different that FANCJ, which can function as both a monomer and dimer [49].

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