Studies of Recq from Deinococcus Radiodurans and Its

Solution Small Angle X-ray Scattering (SAXS) Studies of RecQ from Deinococcus radiodurans and Its Complexes with Junction DNA Substrates*□S Received for publication, July 16, 2013, and in revised form, September 14, 2013 Published, JBC Papers in Press, September 25, 2013, DOI 10.1074/jbc.M113.502112 Wenjia Wang‡, Haifeng Hou‡, Qian Du§, Wen Zhang¶, Guangfeng Liu‡, Eleonora V. Shtykovaʈ, Jianhua Xu‡, Peng Liu‡1, and Yuhui Dong‡2 From the ‡Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China, the §Department of Plant Sciences, College of Agriculture and Nature Resources, University of Connecticut, Storrs, Connecticut 06269, the ¶Department of Physiology, The University of Hong Kong, Hong Kong SAR 999077, China, and the ʈInstitute of Crystallography, Russian Academy of Sciences, Moscow 117333, Russia

Background: RecQ enzymes are homologous recombination repair relevant proteins. Downloaded from Results: Solution structures of full-length DrRecQ protein and its complexes with DNA substrates are defined. Conclusion: DrRecQ catalyzes dsDNA unwinding and Holliday junction migration in a compact state. Significance: Our models provide novel structural information about the way DrRecQ participates in homologous recombination repair. http://www.jbc.org/

RecQ helicases, essential enzymes for maintaining genome genes have been proved to be associated with cancer suscepti- integrity, possess the capability to participate in a wide variety of bility or premature aging. Defects in WRN, BLM, and RecQ4 DNA metabolisms. They can initiate the homologous recombi- helicases are the causes of Werner syndrome, Bloom syndrome, nation repair pathway by unwinding damaged dsDNA and sup- and Rothmund-Thomson syndrome, respectively (2–4). The press hyper-recombination by promoting Holliday junction severe consequences caused by RecQ defects emphasize the at Bibliothekssystem Universitaet Hamburg on July 6, 2018 (HJ) migration. To learn how DrRecQ participates in the homol- importance of the enzyme in preserving chromatin integrity. ogous recombination repair pathway, solution structures of Normally, DNA damages, including the potentially lethal Deinococcus radiodurans RecQ (DrRecQ) and its complexes double strand breaks, can be repaired by homologous recombi- with DNA substrates were investigated by small angle x-ray scat- nation. The RecQ proteins have been proved to take part in the tering. We found that the catalytic core and the most N-terminal recombination and play at least two roles in cells. First, as ATP- HRDC (helicase and RNase D C-terminal) domain (HRDC1) dependent helicases, they can translocate in the 3Ј-5Ј direction undergo a conformational change to a compact state upon bind- and initiate the RecFOR pathway together with SSB (single- ing to a junction DNA. Furthermore, models of DrRecQ in com- strand binding protein) and RecA proteins (5, 6). Second, they plexes with two kinds of junction DNA (fork junction and HJ) possess the ability to promote ATP-dependent branch migra- were built based on the small angle x-ray scattering data, and tion of HJ3 through regions Ͼ2 kb DNA and thus can act as together with the EMSA results, possible binding sites were pro- suppressors of illegitimate recombination (7–11). posed. It is demonstrated that two DrRecQ molecules bind to the D. radiodurans is one of the most radiation-resistant species opposite arms of HJ. This architecture is similar to the RuvAB com- on the earth. It can survive 7000 gray of ionizing radiation with plex and is hypothesized to be highly conserved in the other HJ only 10% cell deaths, whereas most other organisms can only migration proteins. This work provides us new clues to understand suffer less than a few hundred gray. It has been proved that a the roles DrRecQ plays in the RecFOR pathway. dose as high as 7000 gray crushes its 3.28-Mb genome into 20–30-kb fragments by causing 100–150 double strand breaks (12–14). However, the most universal homologous recombina- RecQ proteins are recombination-specific DNA helicases tion repair initiation machine RecBCD is not found in D. radio- that play critical roles in the maintenance of genome stability durans (15, 16). As a result, instead of a backup pathway as it is across all species (1). Mutations in three of the human RecQ in Escherichia coli, the RecFOR pathway is a main homologous recombination repair way in D. radiodurans (17–19). The most conserved structure of the RecQ family proteins is * This work was supported by the grants from the National Basic Research Program of China (2012CB917203) and the National Natural Science Foun- the catalytic core. Composed of one helicase domain and one dation of China (10979005). □S This article contains supplemental Table 1. 1 To whom correspondence may be addressed: Institute of High Energy Phys- 3 The abbreviations used are: HJ, Holliday junction; DrRecQ, RecQ protein from ics, Chinese Academy of Sciences, 19B YuquanLu, Shijingshan District, Bei- D. radiodurans; SAXS, small angle x-ray scattering; Y-DNA, Y structured DNA; jing 100049, China. Tel.: 86-10-88-23-5998; Fax: 86-10-88-23-3201; E-mail: DrRecQfull, full-length DrRecQ; DrRecQ, D. radiodurans RecQ; DrRecQ610, con- [email protected]. struct with catalytic core and HRDC1; DrRecQ-Y, DrRecQ in complex with 2 To whom correspondence may be addressed: Institute of High Energy Phys- Y-DNA; DrRecQ-HJ, DrRecQ in complex with HJ; Y18-12, Y-DNA with one 18-bp ics, Chinese Academy of Sciences, 19B YuquanLu, Shijingshan District, Bei- double strand and two 12-nt single strands; Y7-6, Y-DNA with one 7-bp double jing 100049, China. Tel.: 86-10-88-23-3090; Fax: 86-10-88-23-3201; E-mail: strand and two 6-nt single strands; SEC, size-exclusion chromatography; MM, [email protected]. molecular mass; NSD, normalized spatial discrepancy.

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RQC domain, it is able to catalyze ATP-dependent helicase activity alone (20). HRDC (helicase and RNase D C-terminal) is another typical domain of RecQ and plays a major role in the DNA binding activity (21). DrRecQ is a special member of the RecQ family proteins. Apart from the most conserved catalytic core domain, it has three successive HRDC domains in its C terminus, whereas other RecQ homologs usually contain only one HRDC domain (22, 23). The special domain distribution of DrRecQ may contribute to the high efficiency of D. radiodurans in repairing damaged DNA. High resolution structures of the three HRDC domains of DrRecQ (22) and its homologous catalytic core from E. coli (20) have been solved separately, providing a near complete FIGURE 1. Separation of DrRecQ sequence into domains and linkers. A, full atomic image of the enzyme. However, the enzyme works as schematic representations of domain architectures of DrRecQ and DrRecQ610. DrRecQfull is composed of catalytic core (blue, residues 1–520) and

a whole to achieve its proper function. Thus, it is important three successive HRDC domains (HRDC1 (magenta), residues 536–610; Downloaded from to investigate the architecture of the full-length protein. HRDC2 (orange), residues 654–729; and HRDC3 (green), residues 751–824), whereas DrRecQ610 is the construct lacking HRDC2 and HRDC3 domains. B, Moreover, structural knowledge of DrRecQ in complexes high-resolution structures of the catalytic core from EcRecQ (Protein Data with Y structured DNA (Y-DNA) and HJ can provide us cru- Bank code 1OYW (20)), which possess a 50% sequence homology with the cial information relevant to its functions. In this investiga- catalytic core in DrRecQ; HRDC1 (W. Wang, H. Hou, Q. Du, W. Zhang, G. Liu, E. V. Shtykova, J. Xu, P. Liu, and Y. Dong, unpublished data); HRDC2 (unpublished tion, SAXS reveals solution architectures of DrRecQ protein

data); and HRDC3 (Protein Data Bank code 2RHF (22)). http://www.jbc.org/ and its complexes with DNA substrates. The novel structural information provides us new clues to understand the overall GE Healthcare) with a loading volume of 1 ml for the last step of mechanisms of the enzyme. purification. The size-exclusion chromatography (SEC) was also used to judge molecular masses (MMs) of the complexes. EXPERIMENTAL PROCEDURES EMSAs—All substrates used for EMSA assays are 5Ј-FAM

Sample Preparation—Full-length DrRecQ (DrRecQfull) and its fluorescently labeled DNA complexes and their sequences are at Bibliothekssystem Universitaet Hamburg on July 6, 2018 truncation mutant with catalytic core and HRDC1 (DrRecQ610) listed in supplemental Table S1. Reaction buffer contained 20 were amplified from the genomic DNA of D. radiodurans by PCR. mM Tris-HCl, pH 7.5, 100 mM NaCl, 4 mM MgCl2,and1mM After digestion with the corresponding restriction enzymes, they DTT. In the assays, 10 nM DNA were added into the mixture were ligated into the pET28a expression vectors. The constructed with indicated concentrations of DrRecQ proteins to analyze plasmids were then transformed into E. coli expression strain the binding affinities, whereas 1 ␮M DNA were used to judge BL21(DE3) gold cells for protein expression. the molar ratios of protein and DNA in the complexes. After The purification procedures for DrRecQfull and DrRecQ610 incubation of 30 min at 4 °C, the reaction systems were resolved were identical. Bacterial cells were grown in LB media to mid- in native polyacrylamide gels with TG buffer (25 mM Tris-HCl, log phase at 37 °C in the presence of 50 ␮g/ml kanamycin. and 192 mM glycine, pH 8.3) at 4 °C for 30 min and then visu- Induction of the culture was then carried out with 0.1 mM iso- alized by Typhoon FLA 7000 (GE Healthcare). Dissociation ␤ propyl -D-thiogalactoside at 16 °C for 20 h. The cell pellet was constant (KD) was calculated as the DrRecQ concentration at resuspended in buffer A (25 mM Tris-HCl, 1 M NaCl, 2 mM which half the available DNA was bound and half was unbound. ␤-mercaptoethanol, and 1 mM PMSF, pH 7.5) and disrupted SAXS Data Collection—Synchrotron SAXS measurements using high pressure cell cracker. The supernatant was then were performed at the European Molecular Biology Laboratory loaded onto a nickel-nitrilotriacetic acid resin column (GE on the storage ring DORIS III (DESY, Hamburg, Germany) on Healthcare) and eluted with buffer B (25 mM Tris-HCl, 100 mM the X33 beamline (24) equipped with a robotic sample changer NaCl, and 250 mM imidazole, pH 7.5). Fractions containing (25) and a PILATUS-1 M detector (DECTRIS, Baden, Switzer- DrRecQ were further purified by an ion exchange column (hep- land). All samples were centrifuged at the speed of 13,000 rpm arin, GE Healthcare), where the protein was eluted as a single for 20 min just before measurements to get rid of aggregations peak at 280 mM NaCl. These fractions were finally purified by and sediments. 2 mM DTT was added into the samples and gel filtration (Superdex 200, GE Healthcare) pre-equilibrated in buffers before measurements to avoid radiation damage. buffer C (25 mM Tris-HCl, 500 mM NaCl, and 2 mM DTT, pH All measurements were carried out in vacuum with expo- 7.5). The final yield of the protein was stored at Ϫ80 °C until it sure times of 2 min in eight 15-s frames to monitor for pos- was used for SAXS measurements. sible radiation damage (no radiation effects were detected). The The next step is to purify DrRecQ in complexes with Y-DNA scattering intensity I(s) was recorded in the range of the Ͻ Ͻ Ϫ1 ϭ ␲ ␪ ␭ and HJ (DrRecQ-Y and DrRecQ-HJ). Y18-12 (Y-DNA with one momentum transfer, 0.02 s 0.6 Å , where s (4 sin )/ , ␪ ␭ ϭ 18-bp double strand and two 12-nt single strands) and Y7-6 2 is the scattering angle, and 1.5 Å is the x-ray wavelength. (Y-DNA with 7-bp double strand and two 6-nt single strands) were Due to the considerable experimental noise at higher scattering mixed with the purified proteins with a molar ratio of 1:1, whereas angles, only the most informative part of scattering curves HJ was mixed with proteins with a molar ratio of 1:2. The com- between 0.02 ÅϪ1 and 0.2 ÅϪ1 were used for structural analysis. plexes were then concentrated to 10 mg/ml. Afterward, the mix- To exclude concentration dependence, three different concen- tures were passed through a size-exclusion column (Superdex 200, trations of each sample were prepared and measured. The con-

NOVEMBER 8, 2013•VOLUME 288•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32415 Solution Structures of DrRecQ in Complexes with Junction DNA Downloaded from http://www.jbc.org/ at Bibliothekssystem Universitaet Hamburg on July 6, 2018

FIGURE 2. SAXS analysis of DrRecQ610 and DrRecQfull. A, SAXS scattering data from DrRecQ610 (upper panels) and DrRecQfull (lower panels) in solution: 1, experimental data; 2, scattering pattern computed from ab initio model; 3, smooth curve back transformed from the p(r) function and extrapolated to zero vector; 4, scattering pattern computed from the CORAL model; 5, averaged scattering pattern calculated from the optimized models generated by EOM. LgI, relative, relative intensity of scattering pattern in logarithmic form. B, distance distribution functions for DrRecQ610 (curve 1) and DrRecQfull (curve 2). C and D, ab initio and rigid body reconstructions of DrRecQ610 (C) and DrRecQfull (D). Low-resolution envelopes of typical DAMMIN and GASBOR models are shown both separately (upper panels) and superimposed with atomic models determined by CORAL (lower panels). Views are rotated by 90° according to the vertical axis for each model. In this and other figures, domains are colored as described in Fig. 1, and loops are represented as cyan dots. centrations were 2 mg/ml, 4 mg/ml, and 6 mg/ml for proteins; 1 SAXS Data Processing—All SAXS data were processed with mg/ml, 2 mg/ml, and 3 mg/ml for DNA samples; and 1 mg/ml, the program package ATSAS (26). The scattering of buffers 2 mg/ml, and 4 mg/ml for the complexes, separately. No con- were subtracted from that of the samples, and then were extrap- centration dependence and aggregations were observed during olated to zero concentrations using standard procedures and the measurements. program PRIMUS (27). The resultant curves were used for all

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calculations and reconstructions. MMs of the samples were Distance distribution functions p(r) and maximum diameters

obtained from the extrapolated I(0) values in comparison with Dmax of the scattering objects were calculated using indirect the standard BSA sample. Radii of gyration (Rg) were evaluated Fourier transformation and the program GNOM (28). Ͻ 610 within the range of Guinier approximation sRg 1.3 according Three-dimensional reconstructions for DrRecQ , DrRec- full 610 to Equation 1. Q , and DrRecQ -Y7-6 were performed using programs 2 2 DAMMIN (29), GASBOR (30) and CORAL (31). Ten inde- Ϫs Rg I͑s͒ ϭ I͑0͒expͩ ͪ (Eq. 1) pendent runs for each of the program were compared by the 3 program SUPCOMB (32), and those with the lowest normal- TABLE 1 ized spatial discrepancy (NSD; a measure of quantitative simi- Structural parameters calculated from SAXS data larity among sets of three-dimensional points) were chosen as

Sample MMsequence MMSAXS Rg Dmax typical models. Considering the flexibilities of proteins, pro- kDa kDa Å Å gram EOM (33) was also used to analyze the three specimens 610 Ϯ Ϯ Ϯ DrRecQ 67.4 70.5 2.0 32.0 0.2 110 2 with assemblies of different conformers. The Rg and Dmax dis- DrRecQfull 89.9 93.2 Ϯ 3.5 45.7 Ϯ 0.6 160 Ϯ 2 610 Ϯ Ϯ Ϯ tributions reflected the status of the specimens in solution. For DrRecQ -Y7-6 75.8 79.5 1.5 30.4 0.2 100 2 full 610 full Ϯ Ϯ Ϯ Downloaded from DrRecQ -Y7-6 98.3 96.7 2.5 45.9 0.4 165 2 DrRecQ -Y18-12 and DrRecQ -Y18-12, multiphase models 610 Ϯ Ϯ Ϯ DrRecQ -Y18-12 86.8 84.6 1.0 36.4 0.1 135 2 full Ϯ Ϯ Ϯ were built using program MONSA (29). The program can read DrRecQ -Y18-12 109.3 112.3 2.0 46.1 0.3 170 2 DrRecQ610-HJ 169.8 180.5 Ϯ 1.5 51.8 Ϯ 0.1 180 Ϯ 2 multiple data sets, including not only the scattering from com- full DrRecQ -HJ 214.8 225.9 Ϯ 2.0 56.7 Ϯ 0.2 200 Ϯ 2 plex but also the scattering from DNA and protein alone, http://www.jbc.org/ at Bibliothekssystem Universitaet Hamburg on July 6, 2018

610 full FIGURE 3. Size and Rg distributions of the optimized ensembles for DrRecQ and DrRecQ analyzed by program EOM. A and B represent the 610 full 610 distributions of Dmax (left) and Rg (right) for DrRecQ and DrRecQ , respectively. C, two typical models of DrRecQ in a closed state (selected from the peak with smaller Rg and Dmax, left) and in an open state (selected from the peak with bigger Rg and Dmax, right).

NOVEMBER 8, 2013•VOLUME 288•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32417 Solution Structures of DrRecQ in Complexes with Junction DNA

FIGURE 4. DNA binding affinities of DrRecQ to DNA substrates. Shown is a fraction of Y-DNA (A), HJ (B), or single-stranded DNA (ssDNA) (C) bound to DrRecQ (or its deletion constructs) as a function of protein concentration. Squares, DrRecQfull; circles, DrRecQ610; triangles, catalytic core; inverted triangle, HRDC1. Data Downloaded from were fit using linear regression analysis. KD values are listed for each DrRecQ variant within each plot. http://www.jbc.org/ at Bibliothekssystem Universitaet Hamburg on July 6, 2018

FIGURE 5. DrRecQ forms stable complexes with Y-DNA and HJ. A and B, SEC analysis of DrRecQ-Y18-12 (A) and DrRecQ-HJ (B). C and D, increasing concentrations of DrRecQ (upper panels, DrRecQ610; lower panels, DrRecQfull) bind to 1 ␮M DNA (C, Y-DNA; D, HJ). Numbers above the gel represent the ratios of DNA and protein.

thereby stimulates the scattering objects with different electron domain from E. coli. Below, we present SAXS data for DrRecQ densities to fit scattering data from both monomer and com- by itself and in complexes with Y-DNA and HJ substrates. plex. Several independent runs gave reproducible results from Solution Structures of DrRecQ610 and DrRecQfull—The SAXS which the two with the lowest NSD compared with other mod- profiles for DrRecQ610 and DrRecQfull are shown in Fig. 2A. 610 els were chosen as typical models. Models of DrRecQ -HJ and MMs of the two proteins calculated from SAXS data are prac- full DrRecQ -HJ were built using programs DAMMIN and tically identical to the theoretical values calculated from the SASREF (34), and the most typical DAMMIN model was super- known sequences (Table 1), indicating well behaved, monodis- imposed on the protein phase of SASREF model. perse status of the two specimens in solution. The distance distribution functions for DrRecQ610 and DrRecQfull are shown RESULTS in Fig. 2B. The asymmetrical bell-shaped functions are charac- Experimental Strategy—SAXS data were collected for two teristic for elongated shapes with cross-sections of ϳ40 Å and protein species, the full-length DrRecQ and the construct with- maximum diameters of ϳ110 Å for DrRecQ610 and 160 Å for out HRDC2 and HRDC3 domains (Fig. 1A). Fig. 1B gives DrRecQfull. descriptions of the high resolution structures for each domain To obtain more specific structural information, ab initio of DrRecQ, including a highly homologous catalytic core modeling was applied using the programs DAMMIN and

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GASBOR. Ten independent models generated with both algo- The model of DrRecQ610 (Fig. 2C, lower panels) reveals the ϭ 610 rithms gave reproducible results (NSDav 1.25 for DrRecQ HRDC1 domain arranged slightly apart from the catalytic core. ϭ full full and NSDav 1.78 for DrRecQ , Table 1) and demonstrated good The model of DrRecQ (Fig. 2D, lower panels) demonstrates approximations to the experimental data with discrepancy values the HRDC2 domain distributed far away from the catalytic core chi2 ϭ 1.36 for DrRecQ610 and chi2 ϭ 1.39 for DrRecQfull. The and HRDC1, with the extension of long loop between HRDC1 final models display an ellipsoidal shape for DrRecQ610 (Fig. 2C, and HRDC2. However, instead of a continuous extending out- upper panels) and an elongated shape for DrRecQfull (Fig. 2D, ward, the HRDC3 domain tends to fold back to the catalytic upper panels), consistent with their p(r) functions. core. Importantly, the CORAL models are in good agreement Furthermore, rigid body modeling was applied using the with the ab initio reconstructions as demonstrated by program available high resolution structures and the program CORAL. SUPCOMB (NSD ϭ 1.15 for DrRecQ610 and NSD ϭ 1.68 for DrRecQfull). Ab initio and rigid body modeling methods gave consistent results, showing a reliable averaged overall shapes of the two proteins in solution. There is a 15-residue linker between the catalytic core and

HRDC1 domain in DrRecQ, which is highly conserved in RecQ Downloaded from homologs. In addition, DrRecQ has a unique architecture with two more HRDC domains, which are linked with a 43-residue linker and a 21-residue linker, respectively. The flexibility of these loops might lead to different conformations of the proteins in solution. As a result, we also use an ensemble of con- http://www.jbc.org/ formers to characterize the system. Using the program EOM, a large pool of 10,000 different conformations is generated to analyze the flexibility of the protein, and an optimized ensemble of 50 models that best describes the SAXS data is selected. The selected ensemble of conformations fit the experimental data with chi2 ϭ 0.56 and chi2 ϭ 0.46 for DrRecQ610 and DrRecQfull, respectively. at Bibliothekssystem Universitaet Hamburg on July 6, 2018 610 full The Rg and Dmax distributions of DrRecQ and DrRecQ calculated from the optimized ensemble are shown in Fig. 3, A and B. Two single peaks of the distribution functions imply DrRecQ610 may exist as two distinct conformations in solution: a closed state

(Fig. 3C, left) with a smaller Rg and Dmax, and an open state (Fig. 3C, full right) with a bigger Rg and Dmax. For DrRecQ , the broaden peak means more flexibility of the full-length protein, which probably undergo more continuous conformational changes in solution. full Moreover, the Rg and Dmax distribution functions of DrRecQ have smaller scopes than the ranges of the pools, indicating that FIGURE 6. SAXS analysis of DrRecQ610 in complex with a small junction the full-length protein has limited flexibility and is unable to be DNA substrate. A, scattering profiles and model reconstructions. Curve 1, 610 fully extended in solution. This result is in consistence with the experimental profile of DrRecQ -Y7-6; curve 2, theoretical pattern computed from the ab initio model; curve 3, smooth curve back transformed from the p(r) CORAL model. function and extrapolated to zero angle; curve 4, scattering pattern computed DrRecQ Forms Stable Complexes with Junction DNA—To from the CORAL model; curve 5, averaged scattering pattern calculated from characterize the process of DeRecQ unwinding double- the optimized models generated by EOM. Insets, upper right, monophase 610 models of DrRecQ -Y7-6. a, ab initio model; b, CORAL model; c, superposition stranded DNA and catalyzing HJ migration, it is essential to of the ab initio model and the CORAL model; lower left panel, distance distri- purify stable and monodisperse samples of DrRecQ in com- bution function p(r) for DrRecQ610-Y in solution. LgI, relative, relative inten- 7-6 plexes with Y-DNA and HJ. The DNA binding assays demon- sity of scattering pattern in logarithmic form. B, distributions of Dmax (left) and 610 full 610 Rg (right) for DrRecQ -Y7-6 analyzed by program EOM. strate that DrRecQ and DrRecQ have strong binding TABLE 2 Parameters for SAXS reconstruction models 1 2 610 full Y 18-12 and Y 18-12 represent the Y18-12 DNA binding to DrRecQ and DrRecQ , respectively. DAMMIN or MONSA GASBOR CORAL SASREF Sample ␹ NSD ␹ NSD ␹ NSD EOM (␹) ␹ NSD DrRecQ610 0.55 1.18 1.36 1.25 1.17 1.15 0.56 ϪϪ DrRecQfull 0.63 1.65 1.39 1.78 0.56 1.68 0.46 ϪϪ 610 ϪϪ ϪϪ DrRecQ -Y7-6 0.90 1.14 1.05 0.86 0.61 610 1 ϪϪ ϪϪ Ϫ ϪϪ DrRecQ -Y 18-12 0.79 0.80 full 2 ϪϪ ϪϪ Ϫ ϪϪ DrRecQ -Y 18-12 0.61 0.97 1 ϪϪ ϪϪ Ϫ ϪϪ Y 18-12 0.68 0.95 2 ϪϪ ϪϪ Ϫ ϪϪ Y 18-12 0.80 1.07 DrRecQ610-HJ ϪϪ 1.02 0.97 ϪϪ Ϫ 0.92 1.37 DrRecQfull-HJ ϪϪ 1.06 1.15 ϪϪ Ϫ ϪϪ

NOVEMBER 8, 2013•VOLUME 288•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32419 Solution Structures of DrRecQ in Complexes with Junction DNA Downloaded from http://www.jbc.org/ at Bibliothekssystem Universitaet Hamburg on July 6, 2018

610 610 FIGURE 7. Model reconstructions of DrRecQ in complexes with Y-DNA. A, scattering profiles and MONSA fit for DrRecQ -Y18-12;Y18-12 from DrRecQ - full full Y18-12; DrRecQ -Y18-12; and Y18-12 from DrRecQ -Y18-12 (from top to bottom). LgI, relative, relative intensity of scattering pattern in logarithmic form. B, 610 full 610 normalized distance distribution functions for DrRecQ -Y18-12 (curve 1) and DrRecQ -Y18-12 (curve 2). C and D, multiphase reconstructions for DrRecQ - full Y18-12 (C) and DrRecQ -Y18-12 (D). The reconstructed MONSA models are shown in the upper panels, with the protein phase colored in gray and the DNA phase colored in yellow. The atomic models of proteins are placed inside the corresponding protein-phase envelopes. Views are rotated by 90° according to the horizontal axis for each model.

affinities with Y-DNA and HJ (Fig. 4, A and B) with KD values that one molecule of DrRecQ binds to one molecule of Y-DNA, ranging from 79 to 115 nM. We then mixed DrRecQ with whereas two molecules of DrRecQ bind to one molecule of HJ. Y-DNA and HJ, and the samples were passed through a Super- EMSAs were also taken using 1 ␮M DNA substrates to investi- dex 200 column to check for their homogeneity. DrRecQ-Y and gate the molar ratio of DrRecQ binding to junction DNA. DrRecQ-HJ elute as mono-peaks (Fig. 5, A and B) on the super- Because the DNA concentration is ϳ10 times bigger than the ϭ dex200 column, revealing the attribute of DrRecQ to form sta- KD values of DrRecQ binding to Y-DNA (KD 115 nM) and HJ ϭ ble complexes with junction DNA (either Y-structured fork (KD 105 nM), the fraction of DNA bound increases linearly junction or four-way Holliday junction). MMs calculated from versus the protein concentration. As is shown in Fig. 5, C and D, 610 ϭ full ϭ the elution volumes (VRecQ -Y 14.5 ml, VRecQ -Y 13.3 the DNA substrates can be fully combined to DrRecQ at the 610 ϭ full ϭ ϳ ml, VRecQ -HJ 12.8 ml, VRecQ -HJ 12.6 ml) demonstrate molar ratio of 1:1 for Y-DNA and 1:2 for HJ DNA. This agrees

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well with the SEC results and is further proved by the MMs calculated from the SAXS data (Table 1). DrRecQ Forms a Compact State upon Binding to a Junction DNA—To probe a possible conformational change the catalytic core and the HRDC1 domain undergo when bound to a DNA substrate, SAXS data were collected for DrRecQ610 in complex

with Y7-6. The scattering pattern and the p(r) function of 610 DrRecQ -Y7-6 complex are shown in Fig. 6A. The Rg and Dmax values of the complex calculated from the SAXS data have decreased noticeably as compared with those of the initial protein in solution (Table 1), indicating a more compact status of DrRecQ610 upon binding to the junction DNA. Due to small

MM of Y7-6 (8.5 kDa), monophase modeling changes the final volume insignificantly. Models generated by programs

DAMMIN, GASBOR, and CORAL are in a good agreement Downloaded from with NSD values listed in Table 2. The restorations present the catalytic core and HRDC1 domain being in the closed state (Fig. 6A, insets a–c). To further validate the assumptions, EOM was used to 610 describe the DrRecQ -Y7-6 complex. The ensemble http://www.jbc.org/ selected from the random pool differs from that observed for 610 DrRecQ . Rg and Dmax distributions show single peaks at low values (Fig. 6B), reflecting compact status of DrRecQ610-

Y7-6 in solution. SAXS Reconstructions of DrRecQ in Complex with Fork Junc- tion DNA—To determine the relative position of protein and at Bibliothekssystem Universitaet Hamburg on July 6, 2018 DNA in the DrRecQ-Y complex, SAXS data were also collected 610 full for DrRecQ and DrRecQ in complexes with Y18-12 (Fig. 7A), i.e. with a larger fork junction substrate. Rg values are ϳ 610 full 36.4 nm and 46.1 nm for DrRecQ -Y18-12 and DrRecQ - Y18-12, respectively. p(r) functions are characteristic for elongated shapes (Fig. 7B and Table 1). Multiphase modeling and program MONSA were used to reconstruct ab initio models that include two phases. Several independent runs gave reproducible results with averaged NSD values of 0.8 for DrRecQ610- full Y18-12 and 0.97 for DrRecQ -Y18-12. Good fits to the experimental profiles of complexes and Y-DNA were obtained with discrepancies (chi2) listed in Table 2. The resultant models for 610 full DrRecQ -Y18-12 and DrRecQ -Y18-12 are shown in Fig. 7, C 610 FIGURE 8. Model reconstruction of DrRecQ in complex with HJ. A, SAXS and D, respectively. For DrRecQ -Y18-12, the protein phase data from DrRecQ610-HJ (top) and DrRecQfull-HJ (bottom): 1, experimental has a Dmax of 100 Å and aligned well with the closed state of data; 2, scattering pattern computed from SASREF model; 3, smooth curve 610 DrRecQ . The DNA phase has a part protruding into the pro- back transformed from the p(r) function and extrapolated to zero scattering vector; 4, scattering pattern computed from the ab initio modeling. Inset, dis- tein phase, and the other part stretched out into the solution. 610 full full tance distribution functions for DrRecQ -HJ (curve 1) and DrRecQ -HJ For DrRecQ -Y18-12, the protein phase also has an elongated (curve 2). LgI, relative, relative intensity of scattering pattern in logarithmic shape. Position and orientation of the DNA phase are similar to form. B, SASREF model of DrRecQ610-HJ (top) and its superposition with full that in DrRecQ610-Y . Importantly, the part stretched out DAMMIN model (bottom). C, DAMMIN model of DrRecQ -HJ. Views are 18-12 rotated by 90° according to the vertical axis for each model. into solution has a direct contact with the protein phase (probably with HRDC3 domain). Our EMSA results demonstrate that DrRecQ has much greater binding affinity with single- catalytic core and HRDC1 domain must work together to form ϭ Ͼ stranded DNA (KD 10 nM, Fig. 4C) than with dsDNA (KD a stable complex with the junction DNA, and each domain pro- 2.5 ␮M, data not shown). Therefore, the binding site of Y-DNA vides a binding site for the substrate. should reside in the single stranded regions of DNA. According SAXS Reconstructions of DrRecQ in Complex with Holliday to the analysis above, we deduced that the part protruding into Junction DNA—We then used SAXS to determine the spatial

the protein phase is the single stranded part of Y18-12, whereas organization of DrRecQ-HJ complex (Fig. 8A). SEC results the part stretched out into solution is the double stranded part present DrRecQ610-HJ and DrRecQfull-HJ as single species with

of Y18-12. Moreover, a single catalytic core or a single HRDC exclusion volumes of 12.8 and 12.6 ml, respectively. The corre- domain was shown to have very low binding affinities to junc- sponding MMs are ϳ175 kDa for DrRecQ610-HJ and ϳ220 kDa Ͼ ␮ full tion DNA (KD 2.5 M, Fig. 4). Thus, it is assumed that the for DrRecQ -HJ. These values are significantly greater than

NOVEMBER 8, 2013•VOLUME 288•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32421 Solution Structures of DrRecQ in Complexes with Junction DNA

FIGURE 9. Comparison of DrRecQ and RuvAB promoting HJ migration. A, SAXS model of DrRecQ610 in complex with a HJ. B, schematic drawing of RuvAB complex binding to HJ (RuvA and RuvB are colored in cyan and yellow, respectively). C, schematic diagram of enzymes catalyzing HJ migration. Upper and lower panels represent two rotary views of HJ.

that of the protein monomers. The Rg and Dmax values of the can be considered as a partly unwound dsDNA. Its stable com- Downloaded from two complexes are also larger than those obtained from the plex with DrRecQ gives a good description of a single snapshot protein monomers (Table 1). Taking the SEC and SAXS results of the unwinding process, and therefore provides us clues to into account, we inferred that two DrRecQ molecules bind to understand the way DrRecQ initiates the RecFOR pathway. the HJ. The catalytic core and the HRDC1 domain are highly conserved Because DrRecQ610 forms a rigid compact mode upon bind-

domains in RecQ family. They share a common feature that the http://www.jbc.org/ ing to a junction DNA, the method of molecular tectonics and two domains are linked via a flexible loop. We assume that the program SASREF were applied to model spatial configuration observed binding mode of DrRecQ610 with the Y-DNA sub- 610 of the complex using DrRecQ and HJ DNA as subunits. A strate along with the conformational changes that occur during typical model selected from 10 independent runs demonstrated the interaction may be common in the entire RecQ family pro- 610 that the two DrRecQ subunits consistently bind to the oppo- teins which contain the two domains (E. coli RecQ, Saccharo- site arms of HJ (Fig. 8B, upper panels). Furthermore, ab initio myces cerevisiae Sgs1, human BLM, and WRN, and etc.). at Bibliothekssystem Universitaet Hamburg on July 6, 2018 models were computed using programs DAMMIN and GAS- It should be noted that although the full-length protein is BOR. The final low resolution ab initio model has good self- flexible, it cannot be fully extended in solution. The HRDC3 consistency and superimposes well with the protein phase of domain tends to fold back to the catalytic core and has a direct the SASREF model (NSD ϭ 1.37, Fig. 8B, lower panels). For full contact with the double stranded part of Y-DNA. This kind of DrRecQ -HJ, although rigid body modeling is not applica- domain distribution is consistent with the previous assump- ble due to the high flexibility of the full-length protein, ab tions about HRDC3. It is important for inter-domain interac- initio models demonstrate elongated shapes similar to the tions and is able to help regulate structure-specific DNA bind- DrRecQ610-HJ complex (Fig. 8C), indicating close architec- ing of DrRecQ (22, 23). HRDC3 is also known to contain large tures of the two complexes in solution. negatively charged areas on its surface. Its presence reduces the DISCUSSION binding affinity of DrRecQ to most of DNA substrates. Based on these two points and the SAXS models, we deduce that the Here, SAXS was used to obtain solution structures of full- HRDC3 domain can regulate the DNA binding metabolism by length DrRecQ enzyme and its complexes with junction DNA substrates. These structures give novel insights into the archi- contacting directly with part of the DNA substrate. tectures of the whole enzyme and its binding to DNA sub- The model of DrRecQ-HJ complex reveals that two mole- strates. For the first time, we show that catalytic core and cules of DrRecQ bind to opposite arms of HJ in a closed state HRDC1 domain undergo large-scale conformational changes (Fig. 9A). The RuvAB complex is a well known motor machine to a closed state upon binding to a junction DNA. Furthermore, for HJ migration (35–37). In the complex, RuvA forms an octa- the locations of the Y and HJ DNA were revealed, which pro- mer to recognize and fix a HJ, whereas two RuvB hexameric vided insights into the structural bases of DrRecQ in its helicase rings are in charge of exerting a spiral rotation on each encir- and branch migration activities. cled DNA arm (Fig. 9B). Although the specific structures of the The SAXS results indicate that there might be an equilibrium two complexes vary greatly, they share a common feature that between open and closed conformations of DrRecQ610 in solu- the ATP-dependent helicases (RecQ and RuvB) are located tion. When binding to a junction DNA, the equilibrium shifts on the opposite arms of HJ DNA. With such a structure, the almost completely toward the closed state. Although the bind- helicases unwind the two opposite arms of HJ, whereas the ing site of the complex is not directly visible due to low resolu- other two arms anneal automatically through complemen- tion, additional information such as DNA binding assays pro- tary sequence (Fig. 9C). Thus, the symmetrical architecture is vides clues for possible DNA locations. Because the catalytic highly relevant to its function and is hypothesized to be con- core and HRDC1 domain each provides a binding site for the served in other HJ migration enzymes. Moreover, the RuvAB Y-DNA, the open conformation of protein would expose the complex has a 5Ј-3Ј polarity, whereas RecQ has a reversed 3Ј-5Ј binding sites to solution and help the enzyme to recognize and one. The opposite polarity results in the two enzymes catalyz- bind to the DNA substrates. Moreover, the fork junction DNA ing HJ migration in different orientations and makes RuvAB a

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NOVEMBER 8, 2013•VOLUME 288•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32423 Solution Small Angle X-ray Scattering (SAXS) Studies of RecQ from Deinococcus radiodurans and Its Complexes with Junction DNA Substrates Wenjia Wang, Haifeng Hou, Qian Du, Wen Zhang, Guangfeng Liu, Eleonora V. Shtykova, Jianhua Xu, Peng Liu and Yuhui Dong J. Biol. Chem. 2013, 288:32414-32423. doi: 10.1074/jbc.M113.502112 originally published online September 25, 2013

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