Substrate specificity and helicase polarity

In Xenopus extracts, depletion of RECQ4 prevents the subsequent loading of RPA and DNA polymerase α during replication initiation. These results indicate that DNA melting reactions that generate ssDNA at replication origins may be defective in the absence of RECQ4 (Sangrithi et al, 2005; Matsuno et al, 2006). MCM complex is thought to unwind DNA at the origin of replication; however, in vitro studies have shown that MCM complex can only exert an effect upon DNA substrates containing partial ssDNA (You et al, 2003; Bochman and Schwacha, 2008). We have shown that RECQ4 is capable of unwinding a replication fork-like structure (Figure 1E). To determine whether RECQ4 is capable of unwinding additional substrates reminiscent of DNA structures found during replication, we analysed RECQ4 helicase activity on a 61 bp blunt-end duplex DNA containing either a 12-nt bubble, a 4-nt bubble or without a bubble. Unlike most other human RECQ helicases, which require a 3′ single-stranded tail for efficient DNA unwinding, RECQ4 was found to unwind all three DNA substrates (Figure 2D and E), an activity similar to that exhibited by E. coli RecQ helicase (Umezu et al, 1990; Harmon et al, 1999; Harmon and Kowalczykowski, 2001). The ability of RECQ4 to unwind duplex DNA suggests a possible role of RECQ4 in assisting MCM complex in melting DNA at the origin of replication.

To determine the polarity of the helicase activity of RECQ4, we analysed its activity on 3′- and 5′-overhang substrates. As RECQ4 is capable of unwinding blunt-ended duplex DNA (Figure 2D), reactions were carried out for relatively short times (15 min), as prolonged incubation results in the unwinding of both substrates, as observed previously with E. coli RecQ protein (Umezu et al, 1990). As expected, although DNA unwinding was observed with both substrates, RECQ4 showed a preference towards the 3′-overhang structure (Figure 2F, lower panel) compared with the 5′-overhang substrate (Figure 2F, upper panel). These results show that RECQ4 exhibits a 3′ → 5′ polarity.

RECQ4 possesses dual DNA unwinding activities

In addition to the conserved helicase domain, all RecQ family helicases contain a RecQ C-terminal (RQC) domain, with the exception of human RECQ4 protein. The RQC domain has been shown to be important for the enzymatic activities for the RecQ family helicases (for review, Bennett and Keck, 2004). The lack of a RQC domain in RECQ4 allowed us to determine the minimum domain requirement for the helicase activity of RECQ4. For this, we overexpressed and purified three RECQ4 fragments (Figure 3A and B): (1) RECQ4^1–830 containing the N terminus and the SFII helicase domain; (2) RECQ4^450–1208 containing the helicase domain and the C terminus and (3) the helicase domain alone, RECQ4^450–830. We found that RECQ4^1–830 exhibits ATP-dependent helicase activity similar to the full-length protein, whereas RECQ4^450–1208 shows very little unwinding activity (Figure 3C). When compared with the helicase domain alone (RECQ4^450–830), we found that the presence of the N-terminal sequence in RECQ4^1–830 drastically increased the efficiency of DNA unwinding. In addition, we found that the presence of the C-terminal domain had a mild inhibitory effect on the activity of the SFII helicase domain (Figure 3C, compare RECQ4^450–830 and RECQ4^450–1208). These results indicate that the N-terminal region is required for the efficient DNA unwinding activity of the RECQ4 protein.

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Figure 3

Domain analyses of the RECQ4 protein. (A) Schematic diagram of the RECQ4 fragments. The conserved SFII helicase domain is shown in dark grey, whereas the Sld2-like domain is shown in light grey. (B) Visualization of recombinant RECQ4 fragments purified from E. coli by SDS–PAGE and Commassie blue staining. (C) Helicase activities of the RECQ4 fragments (20 nM each) using duplex DNA in the presence of ATP or AMP-PNP. (D) Helicase activities of the RECQ4 fragments (20 nM each) using splayed arm (upper panel) or 12-nt bubble (lower panel). (E) Schematic diagram of the RECQ4 SFII helicase domain with the seven conserved helicase motifs. Walker A motif (GAGKS) and DEAH box belonging to the Walker B motif are shown. Walker A mutant K508R has Lys508 mutated to Arg508, whereas Walker B mutant D605A has Asp605 mutated to Ala605. (F) Helicase activities of the full-length RECQ4 WT, K508R and D605A proteins (20 nM each) using duplex DNA in the presence of ATP or AMP-PNP.

Surprisingly, when RECQ4^1–492, containing only the N-terminal sequence was analysed, we found that this fragment alone showed efficient ATP-dependent DNA unwinding activity (Figure 3C). The DNA unwinding activity observed with RECQ4^1–492 is significantly more efficient than RECQ4^450–830. RECQ4^1–492 was found to possess strong DNA unwinding activity on duplex DNA, similar to the full-length protein, and also unwinds splayed arm (Figure 3D, upper panel) and 12-nt bubble (Figure 3D, lower panel) substrates. In all cases, RECQ4^450–830 exhibited much weaker helicase activity than RECQ4^1–492. These data suggest that the majority of the ATP-dependent DNA unwinding activity observed with the full-length protein may be contributed by the N-terminal domain. If this is the case, mutations in the conserved Walker A and B motifs of the SFII domain should not abolish the helicase activity observed in the full-length protein. To test this, we generated two full-length RECQ4 point mutants: (1) K508R, in which the conserved lysine residue within the Walker A box of the SFII helicase domain important for ATP hydrolysis was converted to arginine, and (2) D605A, in which the conserved aspartate residue within the ‘DEAH' box important for metal ion binding was converted to alanine (Figure 3E). When compared with the WT protein, ATP-dependent DNA unwinding activity could still be detected in both K508R and D605A mutants (Figure 3F). Interestingly, the D605A mutant consistently showed at least a two-fold decrease in helicase activity compared with WT and K508R. These results indicate that this mutation may affect the stability or overall conformation of the full-length protein and indirectly influence the DNA unwinding efficiency of the N terminus domain.

ATP binding and DNA unwinding of the RECQ4 N-terminal domain

Given that the N-terminal ATP-dependent DNA unwinding activity of RECQ4 was unexpected, we next carried out a direct assessment of the ability of this fragment to bind α³²P-labelled ATP using spin-column assays. First, ATP-binding assays were carried out using full-length RECQ1 and RECQ4 proteins, and showed that both proteins were capable of ATP binding (Figure 4A). The addition of 10-fold excess of unlabelled ATP competed away α³²P-ATP binding by the RECQ proteins (data not shown). To control for nonspecific binding, we used BSA protein, and found that no radiolabel was retained. When RECQ4^1–492 and RECQ4^450–830 were subjected to similar analyses, we found that RECQ4^1–492 bound α³²P-ATP as efficiently as the helicase domain-containing fragment, RECQ4^450–830. The amount of bound α³²P-ATP was directly proportional to the amount of RECQ4^1–492, and ATP binding was found to be dependent on the presence of MgCl₂ (Figure 4B). We further confirmed that only one band, corresponding to the RECQ4^1–492 fragment, became radioactively labelled after UV crosslinking with ATP analogue, α³²P-8-azido-ATP (Figure 4C). These results demonstrate that the RECQ4 N-terminal fragment, RECQ4^1–492, is capable of binding ATP.

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Figure 4

Novel ATP-binding domain of the RECQ4 N terminus. (A) Spin-column analysis of the binding of α³²P-ATP by the indicated protein. Following centrifugation, the α³²P-ATP present in the flow through or void volume indicates the amount bound by the indicated protein. (B) Spin-column analysis of the binding of α³²P-ATP by the indicated amounts of RECQ4^1–492 in the presence (open circle) or absence (close circle) of MgCl₂. (C) Visualization of RECQ4^1–492 fragment binding to α³²P-8-azido-ATP after UV crosslinking by autoradiography following SDS–PAGE. (D) Trypsin proteolysis mapping of RECQ4^1–492 in the presence or absence of ATP. Protein fragments were separated on SDS–PAGE followed by western blot analysis using anti-FLAG antibody. (E) DNA strand annealing activities of the RECQ4 full length (FL) and fragments (5 nM each). DNA annealing of ³²P-labelled S1 and unlabelled complementary S2 oligos were carried out using the indicated proteins without ATP (upper panel) or in the presence of ATP (lower panel).

Cofactor binding to a protein often induces a conformational or oligomerization state change. We therefore tested whether the binding of ATP causes structural changes in RECQ4^1–492. Partial proteolysis mapping of RECQ4^1–492 was carried out in the presence or absence of ATP. When RECQ4^1–492 was digested with trypsin, we found that it was at least four-fold more resistant to proteolytic cleavage in the presence of ATP (Figure 4D). These results indicate that ATP binding induces a conformational change in RECQ4^1–492.

It has been shown that the presence of ATP reduces the DNA annealing efficiency of other RECQ helicases (Garcia et al, 2004; Cheok et al, 2005; Machwe et al, 2005; Muzzolini et al, 2007). We therefore analysed the DNA annealing activity of the RECQ4 fragments and found that fragments of RECQ4 containing the N-terminal region exhibited strong DNA annealing activity in the absence of ATP (Figure 4E, upper panel). As observed with other RECQ helicases, ATP reduced the amount of annealed products in all reactions, including those containing the RECQ4^1–492 fragment. This may be due to ATP-induced protein conformational change or activation of the DNA unwinding activity that counteracts strand annealing (Figure 4E, lower panel).

To date, diverged nucleotide-binding motifs, often glycine rich, have been described in proteins involved in a variety of cellular processes. The RECQ4 N-terminal domain contains several glycine-rich clusters potentially important for ATP binding. To further narrow down the potential ATP-binding site located at the N terminus of the RECQ4, we constructed and purified four smaller N-terminal fragments: (1) RECQ4^1–400, (2) RECQ4^100–480, (3) RECQ4^1–240 and (4) RECQ4^220–480 (Figure 5A and B). ATP-binding assays revealed that ATP-binding efficiency is significantly reduced in RECQ4^1–240 compared with other fragments. This result suggests that the ATP-binding site is located within the domain containing amino-acid residues 240–400 (Figure 5C). We next analysed these N-terminal fragments for their DNA unwinding activities. Interestingly, even though RECQ4^100–480 binds ATP as efficiently as RECQ4^1–400, the lack of the first half of the Sld2-like domain (1–99 aa) in RECQ4^100–480 abolished its DNA unwinding activity (Figure 5D). Therefore, even though the Sld2-like domain is not responsible for ATP binding, it is required for efficient DNA unwinding.

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Figure 5

Biochemical analysis of the RECQ4 N-terminal domain. (A) Schematic diagram of the N-terminal fragments. The amino-acid residues covered by each of the fragments are indicated, and the Sld2-like domain is shown in light grey. (B) Visualization of recombinant N fragments purified from E. coli by SDS–PAGE and Commassie blue staining. (C) Spin-column analysis of the binding of α³²P-ATP by the indicated N-terminal fragments. (D) Helicase activities of different RECQ4 N-terminal fragments using duplex DNA. (E) Binding of ³²P-end-labelled duplex oligos by RECQ4 full length and different RECQ4 N-terminal fragments. DNA–protein complex was analysed on 5% native PAGE. (F) Binding of ³²P-end-labelled single-stranded oligos by RECQ4 full length and different RECQ4 N-terminal fragments. DNA–protein complex was analysed on 5% native PAGE.

We next determined whether the lack of DNA unwinding activity in RECQ4^100–480 is due to a decrease of its affinity for DNA. For this, we analysed the DNA-binding activities of the various N-terminal fragments using electrophoresis mobility shift assays. Full-length RECQ4 binds efficiently to the duplex (Figure 5E) and single-stranded (Figure 5F) substrates. Similar results were obtained with either RECQ4^1–492 or RECQ4^1–400. The presence of RECQ4^1–240 results in a small mobility shift, as indicated by the smearing signal seen close to the free DNA. This less stable but detectable association of RECQ4^1–240 to both ssDNA and dsDNA was observed at the higher protein concentration (i.e. 30 nM), indicating that a ds/ssDNA-binding motif is located within the Sld2 domain. This dsDNA interaction by RECQ4^1–240 may also explain the low amount of ATP-independent DNA melting products observed (Figure 5D). On the other hand, RECQ4^100–480 and RECQ4^220–480 fails to bind to dsDNA. These results indicate that the affinity to dsDNA exhibited by the Sld2 domain is crucial for the DNA unwinding activity of the N terminus of RECQ4.

Proteins

All RECQ4 full-length and truncated proteins, except RECQ4^450–830 WT and D605A mutant, containing N-terminal His tag and C-terminal FLAG tag were purified using the same method. Briefly, proteins were expressed in Rosetta (DE3) pLysS cells. Cells were grown at 37°C to a cell density OD600 ~0.4, followed by overnight induction at 16°C with 0.1 mM isopropyl-β-D-thio-galactoside. Cells were harvested by centrifugation and lysed in lysis buffer (50 mM potassium phosphate, pH 8.0, 10% glycerol, 300 mM KCl, 0.5% Triton X-100, 5 mM β-mercaptoethanol, 1 × protease inhibitor cocktail (Roche), 1 mM PMSF and 0.2 mg/ml lysozyme), followed by sonication. The supernatant was clarified by centrifugation and applied to High-Q column, and the flow through was collected and brought to 0.1% (w/v) in polyethyleneimine (PEI). Precipitate was removed by centrifugation, and the supernatant was applied to Ni-NTA column, washed with buffer B (50 mM potassium phosphate, pH 6.0, 10% glycerol, 500 mM KCl, 0.5% Triton X-100 and 5 mM β-mercaptoethanol) containing 50 mM imidazole, and the His-tagged proteins were eluted with buffer B containing 1 M imidazole. The eluate was dialysed against buffer D (50 mM Tris–HCl, pH 7.6, 10% glycerol and 500 mM KCl) and incubated with 80 μl α-FLAG M2 beads (Sigma) overnight at 4°C. The M2-bound proteins were washed with buffer E (50 mM Tris–HCl, pH 7.6, 10% glycerol, 500 mM KCl, 0.2% Triton X-100 and 1 mM EDTA), eluted with buffer E containing 200 μg/ml FLAG peptide, and dialysed against buffer F (50 mM Tris–HCl, pH 8.0, 10% glycerol, 500 mM KCl, 1 mM EDTA and 1 mM DTT).

E. coli lysates expressing RECQ1, RECQ4^450–830 WT or D605A mutant proteins were first passed through High-Q column followed by PEI precipitation as described for RECQ4. The flow through from Q column was applied onto Ni-NTA column as described, except that 5 mM imidazole was included in the binding buffer. The Ni-NTA eluate was dialysed against buffer G (50 mM Tris–HCl, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM DTT, 0.2% Trion X-100 and 70 mM KCl) and then loaded onto SP column. The SP column was washed with buffer G containing additional 30 mM KCl and eluted with buffer F. All purified proteins were stored as aliquots at −80°C.

DNA substrates

All DNA substrates were prepared by annealing appropriate oligos together and purified on native PAGE. The splayed-arm substrate was generated by annealing ³²P-labelled S1 oligo (5′-GACGCTGCCGAATTCTACCAGTGCCTTGCTAGGACATCTT TGCCCACCTGCAGGTTCACCC-3′) and unlabelled S2 oligo (5′-ATCGATAGTCGGATCCTCTAGACAGCTCCATGTAGCAAGG CACTGGTAGAATTCGGCAGCGT-3′). Blunt-ended duplex was made by annealing ³²P-labelled S1 oligo and unlabelled S1c oligo (5′-GGGTGAACCTGCAGGTGGGCAAAGATGTCCTAGCAAGGCA CTGGTAGAATTCGGCAGCGTC-3′). DNA bubble (12 nt) was made by annealing ³²P-labelled S1 oligo and unlabelled S1BU12 oligo (5′-GGGTGAACCTGCAGGTGGGCAAAGAGTCAGTCAGTCAGCA CTGGTAGAATTCGGCAGCGTC-3′). DNA bubble (4 nt) was made by annealing ³²P-labelled S1 oligo and unlabelled S1BU4 oligo (5′-GGGTGAACCTGCAGGTGGGCAAAGATGTCAGCTCAAGGCA CTGGTAGAATTCGGCAGCGTC-3′). 3′ overhang was made by annealing ³²P-labelled S2c5 oligo (5′-CATGGAGCTGTCTAGAGGATCCGACTATCGA-3′) and unlabelled S2 oligo. 5′ overhang was made by annealing³²P-labelled S1c5 oligo (5′-TGGGTGAACCTGCAGGTGGGCAAAGATGTCC-3′) and unlabelled S1 oligo. The sequence of S4 used as control oligo in the helicase reaction is 5′-AAGGTTCGAATCCTTCCCCCCCCACCACCCCCTCCCCCTC GGCCGAAATTCGGTACC-3′.

Helicase, DNA annealing and DNA-binding assays

Helicase reactions (10 μl) contained ³²P-end-labelled substrate (0.2 ng) in helicase buffer (30 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM ATP, 1 mM DTT, 100 μg/ml BSA and 10% glycerol). Unlabelled S1 (2.5 ng) was included in all the reactions except those experiments shown in Figure 1C and E. Where indicated, 5 mM ATP was replaced by 5 mM AMP-PNP. The reactions were initiated by the addition of purified RECQ proteins and incubated for 30 min at 37°C. The DNA products were deproteinized and analysed on neutral PAGE. Annealing reactions (10 μl) were carried out by incubating the corresponding purified proteins with 10 pg of ³²P-labelled S1 and 25 pg of unlabelled S2 in annealing buffer (30 mM Tris, pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 100 μg/ml BSA and 5 mM ATP where indicated). The reactions were incubated at 37°C for 30 min, deproteinized and analysed on neutral PAGE. DNA-binding reactions (10 μl) were carried out by incubating the corresponding purified proteins with 4 pg of ³²P-end labelled dsDNA or ssDNA in DNA-binding buffer (30 mM HEPES, pH 7.5, 1 mM DTT and 100 μg/ml BSA). The reactions were incubated on ice for 15 min and analysed on neutral PAGE.

ATP-binding assays

For ATP-binding assays using spin columns, ATP-binding reactions (20 μl) were carried out in the presence or absence of MgCl₂ in a reaction buffer (30 mM Tris, pH 7.5, 50 mM KCl, 1 mM DTT and 10% glycerol) plus 1 μCi α³²P-ATP (3000 Ci/mmol, NEN) for 30 min on ice. Reactions were then applied to Bio-Spin P30 Tris chromatography columns (Bio-Rad), which had been pre-equilibrated in a reaction buffer, and spun (4 min at 1000 g) to allow protein with bound α³²P-ATP to pass through. The flow throughs were spotted and dried on Whatman paper and the radioactivity was quantified by phosphorimager. For crosslinking RECQ4^1–492 protein fragment to ATP, 0.4 μM of RECQ4^1–492 fragment was incubated with 10 μM α³²P-8N₃ATP (1 μCi; ALT BioScience) in a binding buffer (20 mM Tris, pH 7.5, 4 mM MgCl₂ and 1 mM DTT). Incubations were performed for 20 min on ice in 10 μl of final volume. Samples were then exposed to 600 mJ/cm² UV radiation and analysed on SDS–PAGE following autoradiography.

This study was supported by grants from American Federation for Aging Research and Yale Rudolph J Anderson Endowed Fellowship. We thank Rebecca L Barnes for her contribution to the initial plasmid cloning.