The Q Motif Is Involved in DNA Binding but Not ATP Binding in ChlR1 Helicase

<div><p>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 <i>ChlR1</i> (<i>DDX11</i>) 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.</p></div

ATP hydrolysis and ATP binding assays of ChlR1 proteins.

<p>(<b>A</b>) A representative image of ChlR1 ATP hydrolysis detected by TLC. (<b>B</b>) ATP binding by ChlR1 proteins was determined by ATP agarose (Jena Bioscience) as described in “Materials and methods”, followed by Western blot with an anti-FLAG antibody. (<b>C</b>) ATP binding by wild-type ChlR1 and mutant protein. α³²P-ATP binding to ChlR1-WT and ChlR1-Q23A was performed by gel filtration chromatography as described in “Materials and methods”. The same amount of protein was used, and the total amount of bound ATP was divided by protein and presented as fmol ATP per pmol protein. BSA was used as a control. (<b>D</b>) A representative image of filter dot blot assays of ChlR1 proteins binding α³²P-ATP. (<b>E</b>) Quantitative analyses of ATP bound to ChlR1 proteins in panel D. Data represent the mean of at least three independent experiments with SD indicated by error bars.</p

Purification and identification of ChlR1 proteins.

<p>(<b>A</b>) ChlR1 proteins (WT and Q23A) were purified and electrophoresed on SDS-polyacrylamide gel and stained with Coomassie blue. (<b>B-C</b>) Western-blot analysis of the purified proteins with antibodies ChlR1 (<b>B</b>) and FLAG (<b>C</b>).</p

Helicase analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.

<p>Helicase reactions were performed by incubating with indicated protein concentration and 0.5 nM duplex DNA substrate (<b>A-B</b>) or OX-1 G2’ DNA substrate (<b>C-D</b>) at 37°C for 20 min. The triangle indicates heat denatured DNA substrate control.</p

DNA binding analysis of ChlR1 proteins on forked duplex DNA and G4 DNA.

<p>The indicated concentrations of ChlR1 proteins were incubated with 0.5 nM forked duplex DNA substrate (<b>A-C</b>) or OX-1 G2’ DNA substrate (<b>D-E</b>) at room temperature for 30 min under standard EMSA conditions as described in “Materials and methods”. The DNA-protein complexes were resolved on native 5% polyacrylamide gels. (<b>F</b>) Variation of fluorescence anisotropy as a function of ChlR1 protein concentration. (<b>G</b>) A representative image of filter dot blot assays of ChlR1 proteins binding forked duplex DNA. (<b>H</b>) Quantitative analyses of DNA bound to ChlR1 proteins in panel G. Data represent the mean of at least three independent experiments with SD indicated by error bars.</p

Thermal stability assays and partial proteolysis mapping of ChlR1 proteins.

<p>(<b>A</b>) Unfolding curves of ChlR1-WT and ChlR1-Q23A over a temperature range from 25 to 60°C. (<b>B</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.</p

Determination of ChlR1 protein oligomerization state.

<p>(<b>A</b>) Coomassie blue stained SDS-PAGE gel showing the ChlR1-WT protein. (<b>B</b>) Chromatographic profiles of ChlR1-WT protein from a HiPrep 16/60 Sephacryl S-300 HR column. (<b>C</b>) Chromatographic profiles of standard proteins on a HiPrep 16/60 Sephacryl S-300 HR column. The equation of protein molecular weight is shown in the upper right corner. (<b>D</b>) Fourteen fractions were selected from the peak area and analyzed by 10% SDS-PAGE. The gel was stained with Coomassie blue. (<b>E</b>) The fractions in D were immunoblotted with an anti-FLAG antibody. (<b>F</b>) Total protein before size exclusion chromatography (SEC), and fractions 4 and 5 after SEC, were subjected to helicase assay using 0.5 nM duplex DNA substrate.</p

ChlR1-Q23A fails to unwind DNA triple helixes.

<p><b>(A-B)</b> Helicase reactions (20 μL) were performed by incubating the indicated ChlR1-WT (<b>A</b>) or ChlR1-Q23A (<b>B</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.</p

SDS-PAGE analysis of ChlR1 proteins by sucrose gradient fractions.

<p>Coomassie blue-stained gels of protein standards (<b>A</b>), and silver staining of ChlR1-WT (<b>B</b>) and ChlR1-Q23A (<b>C</b>) 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.</p

Association between the <i>STK15</i> F31I Polymorphism and Cancer Susceptibility: A Meta-Analysis Involving 43,626 Subjects

<div><p>The association between the Serine/threonine kinase 15 (STK15) F31I polymorphism (rs2273535) and cancer susceptibility remains controversial. To further investigate this potential relationship, we conducted a comprehensive meta-analysis of 27 published studies involving a total of 19,267 multiple cancer cases and 24,359 controls. Our results indicate statistical evidence of an association between the <i>STK15</i> F31I polymorphism and the increased risk of overall cancer in four genetic models: AA vs. TA+TT, AA vs. TT, AA vs. TA, and A vs. T. In a stratified analysis by cancer type, there was an increased risk of breast cancer in four genetic models: AA vs. TA+TT, AA vs. TT, AA vs. TA, and A vs. T, as well as esophageal cancer in two genetic models: AA vs. TA+TT and AA vs. TA. In a stratified analysis by ethnicity, there was a significant increase in cancer risk among Asians, but not Caucasians, in four genetic models: AA vs. TA+TT, AA vs. TT, AA vs. TA and A vs. T. In addition, a stratified analysis by ethnicity in the breast cancer subgroup revealed a significant increase in cancer risk among Asians in two genetic models: AA vs. TA+TT and AA vs. TT, as well as among Caucasians in one genetic model: AA vs. TA. In summary, this meta-analysis demonstrates that the <i>STK15</i> F31I polymorphism may be a risk factor for cancer.</p> </div