Biochemical characterization of Warsaw breakage syndrome helicase

Abstract

Mutations in the human ChlR1 gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in sister chromatid cohesion and hypersensitivity to agents that induce replication stress. A role of ChlR1 helicase in sister chromatid cohesion was first evidenced by studies of the yeast homolog Chl1p; however, its cellular functions in DNA metabolism are not well understood. We carefully examined the DNA substrate specificity of purified recombinant human ChlR1 protein and the biochemical effect of a patient-derived mutation, a deletion of a single lysine (K897del) in the extreme C terminus of ChlR1. The K897del clinical mutation abrogated ChlR1 helicase activity on forked duplex or D-loop DNA substrates by perturbing its DNA binding and DNA-dependent ATPase activity. Wild-type ChlR1 required a minimal 5' single-stranded DNA tail of 15 nucleotides to efficiently unwind a simple duplex DNA substrate. The additional presence of a 3' single-stranded DNA tail as short as five nucleotides dramatically increased ChlR1 helicase activity, demonstrating the preference of the enzyme for forked duplex structures. ChlR1 unwound G-quadruplex (G4) DNA with a strong preference for a two-stranded antiparallel G4 (G2') substrate and was only marginally active on a four-stranded parallel G4 structure. The marked difference in ChlR1 helicase activity on the G4 substrates, reflected by increased binding to the G2' substrate, distinguishes ChlR1 from the sequence-related FANCJ helicase mutated in Fanconi anemia. The biochemical results are discussed in light of the known cellular defects associated with ChlR1 deficiency.

FIGURE 1.

Purification and analysis of recombinant hChlR1 and hChlR1-K897del for helicase and DNA binding activity. A, schematic of hChlR1 protein. The conserved helicase motifs are indicated by yellow boxes, and the positions of the iron-sulfur domain are indicated in orange. The patient deletion mutant K897del and engineered Walker A box (motif I) ATPase mutant K50R are indicated. B, the purity of the hChlR1-WT, hChlR1-K50R, and hChlR1-K897del recombinant proteins was evaluated by the detected migration of the proteins after SDS-PAGE and Coomassie staining according to their predicted sizes. M, protein marker. C, hChlR1 helicase activity on a forked duplex DNA. Helicase reactions (20 μl) were performed by incubating the indicated hChlR1 protein with 0.5 nm duplex DNA substrate at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Triangle, heat-denatured DNA substrate control. D, DNA binding activity of hChlR1 as detected by gel mobility shift assays. The indicated concentrations of hChlR1 proteins were incubated with 0.5 nm forked duplex DNA substrate on ice for 30 min under standard gel shift assay conditions as described under ”Experimental Procedures.” The DNA-protein complexes were resolved on native 5% polyacrylamide gels. Phosphorimages of typical gels for helicase and DNA binding assays are shown in C and D, respectively. Asterisk denotes 5′-³²P end label.

FIGURE 2.

Binding of ATP by hChlR1 wild-type and mutant proteins. A, flowchart for experimental design. Equal amount of hChlR1 proteins (WT, K897del, and K50R) and BSA were incubated with [α-³²P]ATP under identical conditions, and binding mixtures were analyzed by gel filtration chromatography. Liquid scintillation counting was used to determine the amount of ATP contained in each eluted fraction. BSA was used as a control. B, the indicated protein (230 nm final concentration) was incubated with [α-³²P]ATP, and the total amount of bound ATP was divided by protein (fmol of ATP bound/pmol of protein). Experimental data represent the average of at least three independent experiments with S.D. indicated by error bars. The S.D. for K897Rdel is 0.01 fmol of ATP bound/pmol of ChlR1-K897del, resulting in an error bar that is too small to see. TE, Tris-EDTA.

FIGURE 3.

hChlR1 helicase activity on duplex DNA substrates with increasing 5′ ssDNA tail or 3′ ssDNA tail lengths. A, 19-bp duplex DNA substrates with increasing 5′ tail length were incubated with 2.4 nm hChlR1-WT at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” B, 19-bp duplex DNA substrates containing a 15-nt 5′ tail with increasing 3′ tail length were incubated with 0.005 nm hChlR1-WT at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Typical gels are shown on the left, and quantitative analyses are shown on the right. −, without enzyme; +, with enzyme. Triangle, heat-denatured DNA substrate control. Quantitative data represent the mean of at least three independent experiments with S.D. indicated by error bars. Asterisk denotes 5′-³²P end label.

FIGURE 4.

Analysis of hChlR1 helicase activity on replication fork structures. A, helicase reactions (20 μl) were performed by incubating the indicated hChlR1-WT concentrations with 0.5 nm 5′ flap substrate at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Triangle, heat-denatured DNA substrate control. B, quantitative analysis of data from hChlR1-WT helicase reactions on 5′ flap, 3′ flap, and synthetic replication fork structures is shown. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. Asterisk denotes 5′-³²P end label.

FIGURE 5.

Analysis of hChlR1 helicase activity on Holliday junction and D-loop structures. A, helicase reactions (20 μl) were performed by incubating the indicated hChlR1 concentrations with 0.5 nm HJ substrate (upper panel) or forked duplex DNA substrate (lower panel) at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” B, analysis of HJ DNA binding activity of hChlR1 as detected by EMSA. The indicated concentrations of hChlR1 proteins were incubated with 0.5 nm HJ DNA substrate on ice for 30 min under standard gel shift assay conditions as described under ”Experimental Procedures.” The DNA-protein complexes were resolved on native 5% polyacrylamide gels. C, helicase reactions (20 μl) were performed by incubating the indicated hChlR1 (WT, upper; K897del, lower) protein concentrations with 0.5 nm D-loop DNA substrate that contained an invading strand with a 20-nt 5′ ssDNA tail at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Triangle, heat-denatured DNA substrate control. Asterisk denotes 5′-³²P end label.

FIGURE 6.

hChlR1 catalyzes streptavidin displacement from biotinylated oligonucleotide. A, the indicated concentrations of hChlR1 were incubated with 1 mm ATP and 0.5 nm DNA substrate that had streptavidin bound to the covalently linked biotin moiety residing 52 (upper) or 28 nt (lower) from the 5′-end of the radiolabeled oligonucleotide (supplemental Table S2). M, radiolabeled 65-mer oligonucleotide (supplemental Table S2) marker. B, quantitative analyses of hChlR1 streptavidin displacement assays as shown in A. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. Asterisk denotes 5′-³²P end label.

FIGURE 7.

hChlR1 preferentially unwinds two-stranded antiparallel G2′ G-quadruplex compared with four-stranded parallel G4 substrate. A, helicase reactions (20 μl) were performed by incubating the indicated hChlR1 concentrations with 0.5 nm TP-G4 substrate (upper panel) or forked duplex DNA substrate (lower panel) at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Triangle, heat-denatured DNA substrate control. B, helicase reactions (20 μl) were performed by incubating the indicated ChlR1 concentrations with 0.5 nm TP-G4 substrate (upper) or OX-1-G2′ substrate (lower) at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” M, radiolabeled oligonucleotide marker. C, quantitative analyses of hChlR1 helicase assays as shown in B. Data represent the mean of at least three independent experiments with S.D. indicated by error bars. D, helicase assays were performed by incubating 2.4 nm hChlR1-WT with 0.5 nm OX-1-G2′ DNA substrate (left) or 0.5 nm TP-G4 DNA substrate (right) at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” Lane 1, no enzyme control (NE); lanes 2–5, hChlR1 helicase reactions in the absence or presence of the indicated nucleotide (1 mm); lane 6, ³²P-labeled single-stranded oligonucleotide as marker (M). E, helicase reactions (20 μl) were performed by incubating the indicated hChlR1 (K897del, upper; K50R, lower) protein concentrations with 0.5 nm OX-1-G2′ substrate at 37 °C for 30 min under standard helicase assay conditions as described under “Experimental Procedures.” WT, ChlR1-WT was used a control. M, radiolabeled oligonucleotide marker. Asterisk denotes 5′-³²P end label.

FIGURE 8.

Comparison of hChlR1 binding to OX-1-G2′ and TP-G4 G-quadruplex DNA substrates. A, the indicated concentrations of hChlR1-WT were incubated with 0.5 nm TP-G4 DNA substrate (upper) or OX-1 G2′ DNA (lower) on ice for 30 min under standard gel shift assay conditions as described under ”Experimental Procedures.” The DNA-protein complexes were resolved on native 5% polyacrylamide gels. B, quantitative analyses of gel mobility shift assays shown in A. Data represent the mean of three independent experiments with S.D. indicated by error bars.

FIGURE 9.

hChlR1 is preferentially sequestered by OX-1-G2′ compared with TP-G4. A, flowchart for experimental design. ChlR1-WT (5.2 nm) was preincubated with 0.5 nm radiolabeled TP-G4 or OX-1-G2′ in the absence of ATP prior to simultaneous addition of 1 mm ATP and the indicated concentrations of dT₄₅ oligonucleotide. After an additional incubation period at 37 °C, reactions were quenched and analyzed on native polyacrylamide gels as described under “Experimental Procedures.” B, hChlR1-WT helicase activity on the OX-1-G2′ and TP-G4 substrates was analyzed, and percent control (no dT₄₅) unwinding was determined. Shown are averages of data from at least three independent experiments with S.D. indicated by error bars.