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Originally published In Press as doi:10.1074/jbc.M600097200 on March 9, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12849-12857, May 5, 2006
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Three HRDC Domains Differentially Modulate Deinococcus radiodurans RecQ DNA Helicase Biochemical Activity*Formula

Michael P. Killoran1 and James L. Keck2

From the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706-1532

Received for publication, January 4, 2006 , and in revised form, March 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
RecQ helicases are key genome maintenance enzymes that function in DNA replication, recombination, and repair. In contrast to nearly every other identified RecQ family member, the RecQ helicase from the radioresistant bacterium Deinococcus radiodurans encodes three "Helicase and RNase D C-terminal" (HRDC) domains at its C terminus. HRDC domains have been implicated in structure-specific nucleic acid binding with roles in targeting RecQ proteins to particular DNA structures; however, only RecQ proteins with single HRDC domains have been examined to date. We demonstrate that the HRDC domains can be proteolytically removed from the D. radiodurans RecQ (DrRecQ) C terminus, consistent with each forming a structural domain. Using this observation as a guide, we produced a panel of recombinant DrRecQ variants lacking combinations of its HRDC domains to investigate their biochemical functions. The N-terminal-most HRDC domain is shown to be critical for high affinity DNA binding and for efficient unwinding of DNA in some contexts. In contrast, the more C-terminal HRDC domains attenuate the DNA binding affinity and DNA-dependent ATP hydrolysis rate of the enzyme and play more complex roles in structure-specific DNA unwinding. Our results indicate that the multiple DrRecQ HRDC domains have evolved to encode DNA binding and regulatory functions in the enzyme.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Deinococcus radiodurans is an extremophilic bacterial species with an astonishing ability to survive massive levels of DNA-damaging radiation. The D37 dose of ionizing radiation (amount needed to eliminate colony formation by 63% of cells in a population) for D. radiodurans is 6000 Gy. This amount of radiation induces an estimated 275 double-stranded DNA breaks (DSBs)3 and 3,000 single-stranded (ss) DNA breaks per D. radiodurans genome (1, 2). In contrast, introduction of fewer than 10 DSBs/genome is lethal to Escherichia coli as well as to most other species studied to date (2). The robust radioresistance of D. radiodurans makes the bacterium an excellent model system for examining DNA repair mechanisms.

Although many well known DNA repair genes are conserved in D. radiodurans, others are conspicuous in their absence (3). Perhaps the most surprising of these omissions are recB and recC, which encode proteins that are essential for the primary recombinational repair pathway in many bacteria. In the RecBCD pathway, DSBs are processed by the RecBCD helicase/nuclease to create 3'-ssDNA ends onto which RecBCD loads the recombinase protein RecA (4). RecA, in turn, catalyzes homology-dependent invasion of 3'-ssDNA into double-stranded DNA, which primes recombination-dependent replication and allows for repair of the break. In the absence of an apparent RecBCD pathway, the mechanisms responsible for the remarkable ability to repair DNA damage in D. radiodurans are unclear (3, 5).

One possibility that could resolve this conundrum stems from the identification of an intact RecF-like pathway in D. radiodurans. In recBC-E. coli, the RecF recombinational repair pathway can be activated to initiate homologous recombination, utilizing several proteins to substitute for the intrinsic activities of RecBCD (reviewed in Ref. 4). RecF pathway proteins include the RecQ DNA helicase and RecJ 5'-3'-ssDNA exonuclease that are thought to process DSBs, while the RecF, RecO, and RecR proteins mediate RecA loading onto ssDNA (6-8). Activation of the RecF pathway in E. coli requires mutation of the sbcB gene (encoding Exonuclease I, a 3'-5'-ssDNA nuclease) (9), which is thought to be important for preserving the 3'-ssDNA produced by the combined activities of RecQ and RecJ. In contrast to the missing recB and recC, genes encoding RecF pathway proteins are present in D. radiodurans and an sbcB gene is absent (3). Moreover, expression of E. coli Exonuclease I in D. radiodurans cells makes the cells radiation sensitive (10). Thus, it appears that a RecF-like pathway may perform an expanded role in D. radiodurans DNA repair pathways relative to that in wild-type E. coli. These observations have led to investigations into the structures and functions of D. radiodurans RecF pathway proteins that have thus far focused primarily on RecA (11-13), RecO (14, 15), and RecR (16, 17). In this report we have described the first study investigating the structure and function of the RecQ DNA helicase from D. radiodurans.

RecQ helicases are multidomain genome maintenance enzymes with critical roles in bacteria and eukarya (reviewed recently in Refs. 18 and 19). Beyond their aforementioned roles in bacterial RecF pathways, RecQ proteins are critical factors in eukaryotic DNA replication, recombination, and repair. Mutations in three human recQ genes (BLM, WRN, and RECQ4) greatly compromise genomic integrity and result in clinically distinct diseases (20-22). The conserved structural elements found broadly among RecQ helicases include Helicase, RecQ-conserved (RecQ-Ct), and HRDC regions (Fig. 1) (23). In E. coli RecQ (EcRecQ), the Helicase and RecQ-Ct regions fold as a single structural domain referred to as the "catalytic core", whereas the HRDC region forms a second structural domain (24). Within the catalytic core domain, the Helicase region contains conserved sequence motifs that couple ATP hydrolysis to DNA unwinding and the RecQ-Ct region encodes Zn2+-binding and winged helix subdomains that are thought to be important for structural stability and interactions with DNA and proteins (25-27). HRDC domains have been found in both ribonucleases and helicases, suggesting a broad role in nucleic acid binding (23), and isolated HRDC domains from EcRecQ, Saccharomyces cerevisiae Sgs1, and human WRN all bind DNA, albeit with different structural preferences (28-30). RecQ proteins in which the HRDC domain has been inactivated can usually function as DNA-dependent ATPases and helicases but often have altered DNA binding activities. For example, removal or mutation of EcRecQ HRDC domain produces variants with normal helicase and ATPase activities but with altered structure-specific DNA binding properties (24, 30). Similarly, removal or mutation of the HRDC domain in the human BLM protein greatly weakens its binding to and dissolution of double Holliday Junction (HJ) DNA (31), although HRDC-lacking BLM protein is still able to able to hydrolyze ATP and unwind simpler DNA structures (32). HRDC deletion variants from the S. cerevisiae Sgs1 also retain their basic helicase functions in vitro and, in most contexts, their activity in vivo (33-36).

DrRecQ contains a domain architecture similar to RecQ helicases from other organisms. However, in contrast to most other RecQ proteins, DrRecQ encodes three HRDC domains (HRDC1, HRDC2, and HRDC3, in order from N- to C-terminal) (Fig. 1). In this report, we have described the structure-specific DNA binding, DNA-dependent ATP hydrolysis, and DNA unwinding properties of DrRecQ and of a panel of deletion variants that lack either individual HRDC domains or combinations of these domains. Our results indicate that each DrRecQ HRDC domain has a distinct effect on these activities. HRDC1 is required for high affinity DNA binding and is critical for unwinding HJ DNA. HRDC2 attenuates DrRecQ ssDNA binding but stimulates HJ binding; interestingly, HRDC2 also inhibits DNA unwinding. HRDC3 attenuates ssDNA and HJ DNA binding but appears to have no effect on DNA unwinding. In addition, successive removal of HRDC domains from the DrRecQ C terminus increases its maximal ssDNA-dependent ATP hydrolysis rates. Based on our findings, we propose a model where D. radiodurans RecQ activity is regulated by its HRDC domains.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning, Expression, and Purification of DrRecQ Constructs—D. radiodurans cell culture and genomic DNA isolation were carried out as previously described (37). The recQ gene from D. radiodurans was amplified from strain R1 genomic DNA by the polymerase chain reaction. Products were cloned into the pET28.b bacterial expression vector (Novagen) using NdeI and XhoI restriction sites. Domain variants of DrRecQ were constructed similarly to create pMK102 (encoding residues 1-728, DrRecQ-2HRDC), pMK103 (residues 1-610, DrRecQ-1HRDC), pMK104 (residues 1-519, DrRecQ{Delta}C), pMK105 (residues 518-824, 3HRDC), pMK106 (residues 534-610, HRDC1), pMK107 (residues 654-729, HRDC2), pMK108 (residues 751-824, HRDC3), pMK109 (residues 534-729, HRDC1 + 2), and pMK110 (residues 654-824, HRDC2 + 3). Each plasmid encodes a His6 purification tag and thrombin cleavage site on the N terminus of the DrRecQ variant. The fidelity of all D. radiodurans recQ genes was confirmed by DNA sequencing.

E. coli BL21(DE3) cells transformed with pMK101 (or another DrRecQ variant expression vector) were grown in Luria-Bertani medium (38) supplemented with 50 mg/ml of kanamycin at 37 °C with orbital shaking. Protein expression was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside to cells at mid-log phase (A600 = 0.6). Cells were harvested by centrifugation (10 min at 13,000 x g) after 4 h of induction and frozen overnight at -80 °C. Pellets were resuspended in lysis buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, 1 mM 2-mercaptoethanol, 1 mM phenylmethylsulphonyl fluoride, 10 mM imidazole), lysed by sonication on ice, and clarified by centrifugation (30 min at 28,000 x g). Soluble lysate was loaded onto a Ni2+-NTA column and washed with lysis buffer. His-tagged protein was eluted with lysis buffer adjusted to 250 mM imidazole and dialyzed overnight against lysis buffer lacking imidazole. Protein in the dialysate was cleaved with thrombin to remove the His tag (a Gly-Ser-His sequence remains on the N terminus) and passed over an additional Ni2+ -NTA column to remove remaining contaminants. Unbound protein was concentrated and loaded onto a Sephacryl S-100 size exclusion column in 10mM Tris, pH 8.0, 500 mM NaCl, 10% (v/v) glycerol, and 1 mM 2-mercaptoethanol. RecQ fractions were pooled and concentrated to >50 µM, dialyzed against storage buffer (10 mM Tris, pH 8.0, 500 mM NaCl, 40% (v/v) glycerol, 1 mM 2-mercaptoethanol, and 0.5 mM EDTA), and stored at -20 °C. All protein concentrations were determined by measuring A280 in 6.0 M guanidine-HCl (39), and purity was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Limited Proteolysis of D. radiodurans RecQ—Purified DrRecQ was mixed with chymotrypsin at a 100:1 DrRecQ:protease ratio in 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM 2-mercaptoethanol, 1 mM EDTA at room temperature. Reactions were quenched at 0, 1, 5, 15, or 30 min by the addition of 10 mM phenylmethylsulfonyl fluoride and SDS gel running buffer, stored on dry ice, resolved by 10% SDS-PAGE, and stained with Coomassie Brilliant Blue. Proteolysis fragments were excised from the gel, digested with trypsin, and subjected to MALDI-TOF mass spectrometry (University of Wisconsin Mass Spectrometry facility) for identification of DrRecQ peptides retained in each band. In separate experiments, proteolysis fragments were transferred to polyvinylidene difluoride and subjected to N-terminal sequencing (Medical College of Wisconsin Protein/DNA facility).

Synthetic DNA Substrates—Synthetic oligonucleotides dT28, o18 (5'AAGCACAATTACCCACGC-3'), o30 (5'-GCGTGGGTAATTGTGCTTCAATGGACTGAC-3'), oHolliday1 (5'-GCCGTGATCACCAATGCAGATTGACGAACCTTTGCCCACGT-3'), oHolliday2 (5'-GACGTGGGCAAAGGTTCGTCAATGGACTGACAGCTGCATGG-3'), oHolliday3 (5'-GCCATGCAGCTGTCAGTCCATTGTCATGCTAGGCCTACTGC-3'), oHolliday4 (5'-GGCAGTAGGCCTAGCATGACAATCTGCATTGGTGATCACGG-3'), 3'-fluorescein-labeled o18, 3'-fluorescein-labeled o30, and 5'-fluorescein-labeled oHolliday1 were purchased from MWG Biotech. 3'-OH substrate was created by annealing o30-o18 in equimolar amounts, forming an 18-base pair duplex region with a 12-base 3'-single-stranded extension. HJ substrate was created by annealing equimolar amounts oHolliday1, oHolliday2, oHolliday3, and oHolliday4, which forms a structure with four 20-base pair duplex arms extending from a central four-way junction. All substrates were annealed by boiling for 5 min and allowing the mixture to slow cool for 30 min at room temperature. For fluorescence polarization substrates, 3'-fluorescein-labeled o18 and 5'-fluorescein-labeled oHolliday1 oligonucleotides were incorporated into the 3'-OH and HJ structures and 3'-fluorescein-labeled o30 was used as ssDNA.

DNA Binding Assays—Purified DrRecQ variants were serially diluted in dilution buffer (20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM 2-mercaptoethanol, 1 mM MgCl2, 0.1 g/liter of bovine serum albumin, 4% (v/v) glycerol). Diluted proteins were incubated with 1 nM DNA substrate for 20 min at room temperature in a total reaction volume of 100 µl. The fluorescence polarization of each sample was measured at 25 °C using a Panvera Beacon 2000 fluorescence polarization system with 490 and 535 nm excitation and emission wavelengths, respectively. The fraction of substrate bound in each sample was calculated by setting the anisotropy value of the fluorescein-labeled substrate in buffer alone (values were ~45 millianisotropy (mA) units) to zero and setting the anisotropy value at 10 µM protein concentration (values were ~220 mA) to 100% bound. Reactions were performed in triplicate and are plotted as the average fraction DNA bound in the trials with error bars representing one S.D. Uncertainty in the estimated Kd,app values was calculated by determining the standard deviation of the average Kd,app from three independent trials.


Figure 1
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  		FIGURE 1.
DrRecQ HRDC domain variants and alignment of the EcRecQ and DrRecQ HRDC domains. a, schematic diagram of the major conserved domains of EcRecQ and DrRecQ helicases (helicase, red; RecQ-Ct, green; HRDC, blue) and domain variants described in this report. Residue numbers are given to indicate the first and last residue in each construct. b, sequence alignment of EcRecQ and DrRecQ HRDC domains. Residues within the DrRecQ HRDC domains that are invariant with the EcRecQ HRDC domain are shown in boxes; those found in all four domains are highlighted in gray. Residues involved in ssDNA binding in the EcRecQ HRDC domain (30) are shown in red. The secondary structure of the EcRecQ HRDC domain (30) is shown above the alignment with helices indicated in blue boxes. "X" residues at the end of DrRecQ HRDC1 and HRDC2 indicate the number of residues (in subscript) between the domains.

 
DNA-dependent ATPase Assays—ATPase assays were performed as previously described (24). 0.001-100 nM (molecules) dT28 was used to stimulate the ATPase activity of the DrRecQ variants. Reactions were performed in triplicate; average specific activities and error bars representing one S.D. of the mean are plotted.

Helicase Assays—The 5'-end of oligonucleotides o18 and oHolliday 1 were phosphorylated by T4 polynucleotide kinase with [{gamma}-32P]ATP, annealed to their respective complimentary oligonucleotide(s) as described under "Synthetic DNA Substrates" above, resolved by 12% native PAGE, and isolated by electroelution. DNA substrates were dialyzed against 20 mM Tris, pH 8.0, 50 mM NaCl. DrRecQ variants were incubated with ~1nM substrate for 30 min at room temperature in 20 mM Tris, pH 8.0, 50 mM NaCl, 1 mM 2-mercaptoethanol, 1 mM MgCl2, 1 mM ATP, 0.1 g/liter bovine serum albumin, and 4% (v/v) glycerol. Reactions were quenched with the addition of 11% (v/v) glycerol, 0.29% SDS, and 5 ng of unlabeled o18 or oHolliday1. Products were resolved by 12% native PAGE, dried, and imaged using a Amersham Biosciences Storm 820 Phosphorimager. Gel images were quantified using Image-Quant 5.1 software. The percent of DNA unwound is plotted as the average of three trials, and error bars represent one S.D. of the mean. Half-maximal unwinding is defined as the concentration of protein required to unwind 50% of the substrate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of EcRecQ and DrRecQ HRDC Domains—The prototypical bacterial RecQ domain composition is defined by helicase, RecQ-Ct, and HRDC sequence elements in order from N to C terminus. All but four of the currently identifiable bacterial RecQ protein sequences appear to conform to this general structure (data not shown). The outlying RecQ proteins are differentiated by the presence of multiple HRDC domains at their C termini. These unusual RecQ proteins are found in Rhodobacter sphaeroides (two HRDC domains) as well as in D. radiodurans, Neisseria gonorrhea, and Neisseria meningitidis (three HRDC domains). Although RecQ proteins containing a single HRDC domain appear to use this domain as a DNA substrate specificity determinant, the functions of multiple HRDC domains on RecQ helicases have not been previously investigated.

Comparison of the EcRecQ HRDC domain with the three HRDC domains of DrRecQ reveals similarities among the domains that is strongest for the first DrRecQ HRDC domain and somewhat weaker for its more C-terminal domains (Fig. 1). The EcRecQ HRDC domain shares 65% similarity and 40% identity with DrRecQ HRDC1, 64% similarity and 36% identity with DrRecQ HRDC2, and 59% similarity and 33% identity with DrRecQ HRDC3. Of the ~44 residues connecting HRDC1 and HRDC2 and 21 residues connecting HRDC2 and HRDC3 of DrRecQ, one third are either proline or glycine. A total of 14 residues are invariant among the three DrRecQ and single EcRecQ HRDC domains (Fig. 1b). The side chains from 8 of these residues are non-polar and contribute to the hydrophobic core, and 3 others are glycines that form borders of two {alpha}-helices in the EcRecQ HRDC domain structure (30). The remaining 3 invariant residues (2 arginines and 1 glutamic acid) have charged side chains that map to the surface of the EcRecQ HRDC domain. Mutagenesis of the EcRecQ HRDC domain has identified 6 positively charged or aromatic residues that are important for ssDNA binding (30). In each of the DrRecQ HRDC domains, only a subset of these residues is conserved (Fig. 1b). Therefore, it appears that the multiple HRDC domains of D. radiodurans RecQ are not simple repetitive copies of the EcRecQ HRDC domain and each has distinguishing sequence characteristics that may confer specialized functions.


Figure 2
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  		FIGURE 2.
Limited proteolysis defines the domain structure of DrRecQ. a, lanes 1-4, selected purified DrRecQ variants used in this study. Lanes 5-9, products of limited proteolysis of DrRecQ as a function of time (shown above each lane in minutes). Four higher mobility bands are labeled on the right. Protein molecular mass marker positions are indicated on the left in kDa. b, schematic diagram of peptide fragments identified by mass spectrometry within each band indicated in panel a. Each fragment is shown as a red column under its corresponding position in DrRecQ. The relative intensity of each fragment from mass spectrometry is represented by the column height.

 
Limited Proteolysis Defines the Domain Structure of DrRecQ—To examine the low resolution domain structure of DrRecQ, we performed limited proteolysis on purified DrRecQ. The products formed by limited proteolytic treatment were analyzed by SDS-PAGE and mass spectrometry to identify proteolytically resistant domains in DrRecQ. Gel analysis of the reaction products revealed four major bands that migrated faster than full-length DrRecQ (Fig. 2a, lanes 5-9). The estimated masses of these fragments were 85 kDa (band 1), 69 kDa (band 2), 57 kDa (band 3), and 51 kDa (band 4). MALDI-TOF mass spectrometry analysis of trypsin-derived peptide fragments from excised bands 1-4 indicated that successively fewer C-terminal peptides could be detected in the faster migrating bands (Fig. 2b). In addition, the intensity of the C-terminal-most peptides was diminished in the higher mobility bands. These differences were consistent with proteolytic removal of individual HRDC domains from DrRecQ. More definitive N-terminal sequencing attempts failed to identify the specific protease-sensitive sites, perhaps because of heterogeneity of the fragments (data not shown).

Based on this analysis, DrRecQ variants with successive truncations of each of the HRDC domains were recombinantly produced and purified. The SDS-PAGE migration of these proteins was strikingly similar to the limited proteolysis products (Fig. 2a, lanes 1-9), consistent with our hypothesis that the DrRecQ fragments differ by removal of one or more HRDC domains. Additional recombinant proteins containing each individual DrRecQ HRDC domain (HRDC1, HRDC2, and HRDC3), each pairwise combination (HRDC1 + 2 and HRDC2 + 3), and the triple HRDC sequence (3HRDC) were also purified for biochemical analysis. Circular dichroic spectra and/or structure determination of the single, double, and triple HRDC constructs indicated that the HRDC constructs were folded and highly {alpha}-helical (data not shown).

DNA Binding by DrRecQ Variants—The DNA binding properties of each DrRecQ variant was assessed by measuring its equilibrium binding affinity to three different synthetic DNA substrates: ssDNA, 3'-OH DNA, and HJ DNA. Given the many previous reports of HRDC-mediated DNA binding, the effects of removing the DrRecQ HRDC domains on its ssDNA binding affinity were surprising (Fig. 3a). The apparent dissociation constant (Kd, app) measured for DrRecQ binding to ssDNA was 12 ± 2nM. However, DrRecQ-2HRDC, which lacks HRDC3, had an increased ssDNA binding affinity (Kd,app of 2.8 ± 0.4 nM). Furthermore, DrRecQ-1HRDC, which lacks both HRDC2 and HRDC3, had an even greater enhancement of ssDNA binding affinity relative to full-length protein, with a Kd,app of less than 1 ± 0.2 nM for DrRecQ-1HRDC. An upper limit for Kd,app is given because the binding experiment includes 1 nM DNA and the DrRecQ-1HRDC concentration required for half-maximal binding is ~1nM, making substrate concentration limiting and implying that the true Kd for the binding reaction is below 1 nM. In contrast, removal of all three HRDC domains from DrRecQ (DrRecQ{Delta}C) dramatically decreases affinity for ssDNA as is evident by its 380 ± 50 nM Kd, app. Thus, HRDC2 and HRDC3 appear to inhibit high affinity ssDNA binding, whereas HRDC1 forms a critical ssDNA binding element when linked to the DrRecQ catalytic core domain.


Figure 3
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  		FIGURE 3.
DNA binding activity of DrRecQ is modulated by the presence of its multiple HRDC domains. Fraction of ssDNA (a), 3'-OH (b), or HJ (c) DNA bound to DrRecQ (or a deletion variant) as a function of protein concentration. DrRecQ, closed diamonds; DrRecQ-2HRDC, open triangles; DrRecQ-1HRDC, closed triangles; DrRecQ{Delta}C, closed squares; and 3HRDC, X. Data are the average of three repetitions and were fit using linear regression analysis; error bars reflect one S.D. Apparent Kd values are listed for each DrRecQ variant within each plot. Uncertainty in Kdapp values was calculated as the S.D. of three independent trials. Trend lines were calculated using Curve Expert.

 
To test whether the HRDC domains alone are sufficient for ssDNA binding, we measured the binding affinity of a construct comprising just the three HRDC domains (3HRDC). This construct bound ssDNA with a Kd,app of ~47 ± 1 nM. Interestingly, each of the individual HRDC domains alone and constructs composed of dual HRDC domains bound ssDNA very poorly with Kd,app values in excess of 10 µM (data not shown). Thus, it appears that the DrRecQ catalytic core and HRDC1 combine to form a high affinity ssDNA binding site, whereas the 3HRDC construct retains a modest ssDNA binding ability.

The apparent equilibrium ssDNA binding constants of the DrRecQ variants were somewhat salt dependent (data not shown). Relative to binding data described above (50 mM NaCl), the ssDNA binding affinity of DrRecQ was unaffected by raising the NaCl to 100 mM but was 4-fold weaker in 150 mM NaCl. ssDNA binding by DrRecQ-2HRDC and DrRecQ-1HRDC was 4-10 fold weaker when salt concentrations in the reaction buffer were raised to 100 and 150 mM NaCl. Interestingly, ssDNA binding to DrRecQ{Delta}C was enhanced ~9-fold in 100 mM NaCl relative to 50 mM, but at 150 mM NaCl the affinity was indistinguishable from the 50 mM NaCl data. Thus, with few exceptions, higher salt concentrations reduced the ssDNA binding affinities of the DrRecQ variants.

Examination of binding to a partial duplex DNA substrate with a 3'-ssDNA overhang revealed a different relationship among the panel of DrRecQ variants. Although EcRecQ binds a variety of DNA structures, 3'-OH DNA structures, which mimic the lagging strand of a replication fork, are bound with the highest affinity (40, 41). We therefore examined DrRecQ DNA binding to a simple 3'-OH DNA. DrRecQ, DrRecQ-2HRDC, and DrRecQ-1HRDC had very similar Kd,app values of 49 ± 9, 56 ± 10, and 56 ± 5 nM, respectively, on 3'-OH DNA (Fig. 3b). A noticeable effect on DNA binding was observed, however, upon removal of all three HRDC domains; DrRecQ{Delta}C displayed a 10-fold reduction in binding affinity for the 3'-OH substrate. The 3HRDC construct bound 3'-OH DNA similarly to DrRecQ{Delta}C. Again, each of the individual HRDC domains, HRDC1 + 2, and HRDC2 + 3 had very low affinity for this substrate with Kd,app values in excess of 10 µM (data not shown). These binding data reinforce the idea that HRDC1 is critical for high affinity DNA binding; however, in contrast to ssDNA, HRDC2 and HRDC3 do not negatively affect 3'-OH DNA binding.

Finally, we measured HJ DNA binding affinity for each of the DrRecQ constructs. These DNA structures are common intermediates of recombination reactions, and several RecQ proteins are able to bind HJ DNA and catalyze branch migration reactions (reviewed in Ref. 18). The effects of removing DrRecQ HRDC domains on HJ binding were more complex than observed for ssDNA binding. Full-length DrRecQ binds HJ DNA with a Kd,app of 8.5 ± 3 nM (Fig. 3c). Interestingly, both DrRecQ-2HRDC and DrRecQ-1HRDC exhibited apparent biphasic DNA binding behavior with HJ DNA. DrRecQ-2HRDC binds the substrate with higher affinity than full-length DrRecQ at low protein concentrations but does not reach DNA binding saturation in an apparent two-state binding reaction. DrRecQ-1HRDC binds the substrate with lower affinity than DrRecQ at all protein concentrations tested but also appears to bind HJ DNA in a complex manner. It is possible that DrRecQ-2HRDC and DrRecQ-1HRDC have multiple binding sites on the HJ substrate and interact with either the duplex arms or the fourway junction of the substrate with different affinities. Both 3HRDC and DrRecQ{Delta}C variants showed low affinity for the HJ substrate. As was the case with ssDNA and 3'-OH DNA, the individual HRDC domains, HRDC1 + 2, and HRDC2 + 3 have very low affinity for HJ DNA with Kd,app values in excess of 10 µM (data not shown). Thus, the HRDC domains of DrRecQ play different roles in binding to HJ DNA: HRDC1 and HRDC2 are required for maximal HJ binding by DrRecQ, whereas HRDC3 appears to reduce HJ binding at low protein concentrations.

Comparison of the three DNA substrates tested indicates that DrRecQ has the highest affinity for ssDNA and HJ DNA. Removal of HRDC3 increases the affinity of DrRecQ for these substrates. The presence of HRDC2 weakly decreased DrRecQ binding to ssDNA; however, its loss lowered the affinity for HJ DNA. For all three substrates, HRDC1 is required for high affinity DNA binding, and its absence results in a dramatic decrease in binding affinity. Individually purified HRDC1, HRDC2, and HRDC3 and pair-wise combinations of the domain all bind the substrates with very low affinity (>10 µM).

ATPase Activity of DrRecQ Variants—To test the effects of removing Dr RecQHRDC domains on its ATPaseactivity, we measured the ssDNA-dependent ATPase activity of each variant. DrRecQ DNA unwinding activity precludes using more complex DNA structures as cofactors because ssDNA and double-stranded DNA differentially stimulate RecQ ATPase activity (42). ATPase stimulation studies were therefore limited to using homopolymeric ssDNA (dT28) as a cofactor.

As is the case with other RecQ helicases, each of the constructs required DNA as a cofactor to produce measurable ATP hydrolysis (Fig. 4). Full-length DrRecQ hydrolyzed ATP with a maximal rate of 900 min-1 and is half-activated in the presence of ~1nM dT28; removal of either HRDC3 or both HRDC2 and HRDC3 led to an increase in the maximal ATPase rate to 1620 and 1850 min-1, respectively. The amount of dT28 required for half-activation of the DrRecQ-2HRDC and DrRecQ-1HRDC ATPase activities was ~3 nM. Although we were unable to reach a maximal hydrolysis rate for DrRecQ{Delta}C, it has the highest observed rate for all of the variants with an ATPase rate of 2430 min at 100 nM DNA. Despite this heightened ATPase activity, DrRecQ{Delta}C required higher ssDNA concentrations to stimulate ATPase activity, consistent with its relatively low affinity for ssDNA (Fig. 3).


Figure 4
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  		FIGURE 4.
The maximal rate of ATP hydrolysis is increased by removal of DrRecQ HRDC domains. Specific ATPase rates are plotted as a function of DNA concentration. Representative trend lines are included for clarity. DrRecQ, closed diamonds; DrRecQ-2HRDC, open triangles; DrRecQ-1HRDC, closed triangles; and DrRecQ{Delta}C, closed squares. Data are the average of three repetitions; error bars reflect one S.D.

 
Helicase Activity of DrRecQ Variants—Finally, we examined the DNA unwinding activity of each of the DrRecQ variants. Helicase activity requires the productive integration of DNA binding and ATP hydrolysis activities, which makes it an excellent measure of the overall effects of removing the HRDC domains from DrRecQ. Helicase activity was measured for 3'-OH and HJ DNA substrates. In each case, a single radiolabeled oligonucleotide was incorporated into the DNA substrate and helicase activity was assessed by the mobility of the labeled DNA on non-denaturing PAGE. The annealed DNA substrate displays retarded gel mobility relative to the unwound ssDNA product.

Full-length DrRecQ unwound 3'-OH DNA with half-maximal activity observed at ~0.1 nM enzyme concentration (Fig. 5a and supplemental Fig. S1). Removal of HRDC3 had no apparent effect on the ability of the enzyme to unwind this substrate. However, removal of HRDC2 and HRDC3 stimulated unwinding of 3'-OH DNA ~10-fold over fulllength DrRecQ (Fig. 5a). DrRecQ{Delta}C 3'-OH unwinding activity was indistinguishable from that of DrRecQ-1HRDC.

DrRecQ was also able to unwind a synthetic HJ DNA substrate, albeit with a reduced efficiency relative to 3'-OH DNA unwinding (Fig. 5b). Half-maximal unwinding of HJ DNA by DrRecQ required ~0.3 nM enzyme, 3-fold more than was required for the unwinding of the 3'-OH substrate. As was the case with 3'-OH DNA unwinding, the amount of enzyme required for half-maximal HJ DNA unwinding was unaffected by removal of HRDC3 but was significantly reduced by removal of HRDC2 and HRDC3. Activity of this variant appears to be biphasic, which could be related to its HJ binding behavior (Fig. 3). In contrast to 3'-OH unwinding, removal of all three HRDC domains substantially reduced HJ DNA unwinding such that a concentration of at least 50 nM was required to unwind half of the substrate.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we tested a hypothesis that the multiple HRDC domains in DrRecQ helicase have specialized functions relative to each other and to HRDC domains from well studied single HRDC RecQ proteins. The canonical bacterial RecQ structure combines a catalytic core domain with a single HRDC domain. In contrast, four genes encoding RecQ proteins with multiple HRDC domains have been found through several genome sequencing projects, raising the possibility that the additional HRDC domains present in these RecQ family members encode unique biochemical activities. Sequence comparison among the HRDC domains of D. radiodurans and E. coli suggested that each domain likely shares a similar overall structure due to the high degree of sequence conservation. However, differences in the sequences between EcRecQ and DrRecQ HRDC domains in putative surface residues that form critical ssDNA binding elements in the E. coli enzyme suggest that the DrRecQ domains could have different functions from the currently understood roles of other HRDC domains.


Figure 5
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  		FIGURE 5.
DNA unwinding activity of DrRecQ is modulated by its HRDC domains. Percentage of substrate unwound by each domain variant is shown for 3'-OH (a) and HJ (b) DNA substrates as a function of protein concentration. DrRecQ, closed diamonds; DrRecQ-2HRDC, open triangles; DrRecQ-1HRDC, closed triangles; and DrRecQ{Delta}C, closed squares. Data are the average of three repetitions and were fit using linear regression analysis; error bars reflect one S.D. Trend lines were calculated using Curve Expert.

 
Limited proteolysis experiments indicated that each DrRecQ HRDC domain forms discreetly folded modules in the enzyme (Fig. 2). These observations led to the construction of a series of deletion variants that successively removed each DrRecQ HRDC domain. Using these variants, we found that the multiple HRDC domains of DrRecQ regulate its activity by modulating the enzyme's structure-specific DNA binding affinity, DNA-dependent ATP hydrolysis behavior, and helicase activity.

DrRecQ HRDC Domains Differentially Modulate DNA Binding and Enzymatic Activity—Full-length DrRecQ was shown to bind to HJ and ssDNA with high affinity and to 3'-OH DNA with a somewhat lower affinity (Fig. 3). As is the case with EcRecQ (42, 43), DrRecQ is competent to hydrolyze ATP in a DNA-dependent fashion (Fig. 4) and unwind HJ and 3'-OH DNA structures in an ATP-dependent reaction with 3'-OH being the preferred substrate (Fig. 5). DrRecQ could also unwind 5'-OH, albeit with a significantly reduced activity relative to 3'-OH unwinding (data not shown).

Removal of the C-terminal-most HRDC domain from DrRecQ (HRDC3) resulted in surprising differences in activity relative to fulllength protein. Most strikingly, removal of HRDC3 produced a DrRecQ with ~5-fold higher affinity for ssDNA (Fig. 3) and a heightened maximal ssDNA-dependent ATPase rate (Fig. 4). However, these enhancements did not translate to an increase in DNA unwinding activity (Fig. 5). Together, these data suggest that DrRecQ HRDC3 plays a negative regulatory role that attenuates association of the enzyme with ssDNA. This is in stark contrast to the role played by the EcRecQ HRDC, which directly binds ssDNA (30).

Removal of both HRDC2 and HRDC3 from DrRecQ in the DrRecQ-1HRDC protein resulted in additional functional changes. Removal of HRDC2 further enhanced ssDNA binding relative to DrRecQ-2HRDC, whereas HJ binding was impaired and 3'-OH binding was unaffected (Fig. 3). DNA-dependent ATPase activity was also essentially the same as that of DrRecQ-2HRDC (Fig. 4). Remarkably, DrRecQ-1HRDC displayed the most robust unwinding activity of the variants tested, with ~10-fold higher specific unwinding activity on 3'-OH and HJ DNA relative to full-length DrRecQ (Fig. 5). Taken together, it appears that whereas HRDC3 inhibits ssDNA binding, removal of HRDC2 and HRDC3 stimulates myriad functions in DrRecQ. Thus, the role of DrRecQ HRDC2 is likely more complex than that of HRDC3, with properties that limit both DNA binding and unwinding.

Finally, removal of all three of the DrRecQ HRDC domains in the DrRecQ{Delta}C construct produced a variant with greatly weakened DNA binding ability on all substrates tested (Fig. 3) but that had the highest levels of ssDNA-dependent ATPase rates among the panel of constructs tested (Fig. 4). Interestingly, the weaker DNA binding ability did not result in a change in 3'-OH unwinding capacity relative to DrRecQ-1HRDC, although HJ unwinding is greatly attenuated in the DrRecQ{Delta}C protein (Fig. 5). These observations indicate a positive role for HRDC1 in DNA binding and a regulatory role that restricts ATP hydrolysis in the protein. Collectively, these data suggest that each HRDC domain differentially modulates structure-specific DNA binding and catalytic activities of DrRecQ.

Models of the Functions of Individual DrRecQ HRDC Domains—This report defines novel regulatory roles for DrRecQ HRDC domains. HRDC domain regulation appears to affect both structure-specific DNA binding and enzymatic activity of DrRecQ, in that HRDC2 and HRDC3 generally inhibit and HRDC1 generally stimulates these activities. A simple model to explain our data involves direct contacts between the HRDC domains and the catalytic core domain of DrRecQ. HRDC3 appears to most strongly attenuate ssDNA binding in DrRecQ. It is possible that HRDC3 directly binds a DNA binding site on DrRecQ and competes with ssDNA for binding the enzyme. In support of a competitive model, the calculated pI for HRDC3 is 4.7, indicating that it likely contains a highly electronegative surface that could potentially mimic the negative charges of DNA and compete with DNA for binding. Similar modes of DNA binding inhibition have been observed for several proteins (44). Alternatively, HRDC3 could indirectly inhibit DNA binding by binding to and stabilizing a conformation of the enzyme with lower enzymatic and/or DNA binding activity. In contrast to HRDC3, HRDC2 primarily attenuates DrRecQ DNA unwinding. A simple competition model does not explain its role; ssDNA-dependent ATPase activity is quite similar between constructs that differ by the presence of HRDC2, and its effects on DNA binding are complex. Instead, HRDC2 appears to modulate intrinsic DrRecQ helicase activity, because its removal strongly stimulates unwinding of HJ and 3'-OH DNA without enhancing binding affinity. Regardless of the precise mechanisms whereby the presence of HRDC3 and HRDC2 inhibit DrRecQ activity, it is clear that these domains play negative regulatory roles. Finally, HRDC1 appears important for DNA binding and HJ DNA unwinding, making it a stimulator of DrRecQ activity. This is similar to the observed role of the single HRDC domain in EcRecQ (24, 30). Additional potential roles of DrRecQ HRDC domains include acting as sites for protein-protein interactions that could target the enzyme to damaged DNA structures and/or provide a cellular mechanism for regulating the actions of the HRDC domains on DrRecQ in different contexts.

DNA binding inhibition and regulation of catalytic functions of DrRecQ by HRDC2 and HRDC3 reveal a novel role for these domains in RecQ function. Single HRDC RecQ helicases use their HRDC domains as auxiliary DNA binding element; in DrRecQ, however, its additional HRDC domains have evolved more complex regulatory roles. It follows that regulation by HRDC2 and HRDC3 is likely to be important for DrRecQ function in vivo. This is consistent with the proposed central role of RecQ and a RecF-like recombination pathway in D. radiodurans cells. Genome maintenance models that include HJ and 3'-OH DNA structures as DSB repair intermediates have been suggested to be important in D. radiodurans (2); the studies presented here have demonstrated that the activity of DrRecQ on these structures is regulated by its HRDC domains. It is reasonable to speculate that the DrRecQ HRDC domains aid in substrate selection in vivo and in regulating the level of activity of the helicase in DNA structure-specific manner.

Interestingly, RecQ is not the only genome maintenance protein in D. radiodurans that has an unusual arrangement of domains playing rare regulatory roles. D. radiodurans ssDNA-binding protein (SSB) encodes an atypical domain arrangement with two DNA-binding domains per polypeptide rather than the single domain found in over 99% of other bacterial SSBs (45, 46). This arrangement allows independent evolution of the two domains not available in the single domain SSBs that has been proposed to be important for specialized functions of the two domains in protecting exposed ssDNA (46). Thus, duplication of a domain with subsequent selection of novel functions appears to be an evolutionary strategy that has helped shaped multiple genome maintenance proteins in D. radiodurans.

Parallels with Other Triple HRDC Domain-containing RecQ Helicases—The only other organisms identified thus far that have recQ genes with three HRDC domains are N. meningitidis and N. gonorrhea. How might the studies presented here relate to these other bacterial species? N. meningitidis and N. gonorrhea are obligate human pathogens with a high demand for recombination to facilitate antigenic variation. Antigenic variation in Neisseria is a highly active process that occurs at rates of 10-4 to 10-3 events/cell/generation (47). Even though the Neisseria species do have recBCD homologs, mutation of recQ in N. gonorrhea results in severe reductions in antigenic variation and DNA repair capability (48). This condition is reminiscent of the compromised repair of E. coli recQ mutations in a recBC-sbcB- background (49, 50). This suggests an important role for RecQ and a RecF-like pathway in Neisseria as has been hypothesized for D. radiodurans. Thus, although Deinococcus and Neisseria species inhabit different ecological niches, their similarities suggest they may utilize similar RecQ-dependent strategies of DNA recombinational repair. It could be that these organisms require high fidelity (and highly regulated) homologous recombination reactions and have developed specialized activities for their RecQ proteins to initiate these reactions. As such, the regulatory roles of the DrRecQ HRDC domains described in this report could represent important mechanistic features of multi-HRDC domain enzymes.


   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants GM068061 andGM067085 and by a Shaw Scientist grant (to J. L. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 A Cremer Scholar. Supported in part by a National Institutes of Health training grant in molecular biophysics. Back

2 To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, 550 Medical Science Center, 1300 University Ave., Madison, WI 53706-1532. Tel.: 608-263-1815; Fax: 608-262-5253; E-mail: jlkeck{at}wisc.edu.

3 The abbreviations used are: DSB, double-stranded DNA break; ss, single-stranded;HRDC, helicase and RNaseD C-terminal; DrRecQ, D. radiodurans RecQ; EcRecQ, E. coli RecQ; HJ, Holliday junction; 3'-OH, partial duplex DNA with a 3'-single-stranded overhang; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. Back


   ACKNOWLEDGMENTS
 
We thank Jennifer Fostel and Susan Marqusee for help with CD measurements and members of the Keck laboratory for critical reading of the manuscript.



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 DISCUSSION
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