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• W2115410512 abstract "Article25 January 2007free access Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function Olga Koroleva Olga Koroleva Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Nodar Makharashvili Nodar Makharashvili Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Charmain T Courcelle Charmain T Courcelle Department of Biology, Portland State University, Portland, OR, USA Search for more papers by this author Justin Courcelle Justin Courcelle Department of Biology, Portland State University, Portland, OR, USA Search for more papers by this author Sergey Korolev Corresponding Author Sergey Korolev Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Olga Koroleva Olga Koroleva Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Nodar Makharashvili Nodar Makharashvili Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Charmain T Courcelle Charmain T Courcelle Department of Biology, Portland State University, Portland, OR, USA Search for more papers by this author Justin Courcelle Justin Courcelle Department of Biology, Portland State University, Portland, OR, USA Search for more papers by this author Sergey Korolev Corresponding Author Sergey Korolev Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA Search for more papers by this author Author Information Olga Koroleva1, Nodar Makharashvili1, Charmain T Courcelle2, Justin Courcelle2 and Sergey Korolev 1 1Edward A Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St Louis, MO, USA 2Department of Biology, Portland State University, Portland, OR, USA *Corresponding author. Department of Biochemistry and Molecular Biology, St Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA. Tel.: +1 314 977 9261; Fax: +1 314 977 9205; E-mail: [email protected] The EMBO Journal (2007)26:867-877https://doi.org/10.1038/sj.emboj.7601537 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info RecF, together with RecO and RecR, belongs to a ubiquitous group of recombination mediators (RMs) that includes eukaryotic proteins such as Rad52 and BRCA2. RMs help maintain genome stability in the presence of DNA damage by loading RecA-like recombinases and displacing single-stranded DNA-binding proteins. Here, we present the crystal structure of RecF from Deinococcus radiodurans. RecF exhibits a high degree of structural similarity with the head domain of Rad50, but lacks its long coiled-coil region. The structural homology between RecF and Rad50 is extensive, encompassing the ATPase subdomain and the so-called ‘Lobe II’ subdomain of Rad50. The pronounced structural conservation between bacterial RecF and evolutionarily diverged eukaryotic Rad50 implies a conserved mechanism of DNA binding and recognition of the boundaries of double-stranded DNA regions. The RecF structure, mutagenesis of conserved motifs and ATP-dependent dimerization of RecF are discussed with respect to its role in promoting presynaptic complex formation at DNA damage sites. Introduction DNA damage encountered during replication can generate gaps in newly synthesized DNA, arrest progression of the replication machinery, or lead to breakdown of the replication fork and double-stranded (ds) DNA breaks (DSBs) (Cox et al, 2000). When these events are not accurately repaired, they can result in mutations, genomic rearrangements, or even cell lethality. Recombination proteins play essential roles in maintaining replication in the presence of DNA damage and allowing replication to resume (Kuzminov, 2001; Cox, 2002). In Escherichia coli, the homologous recombination proteins are classified into two predominant RecA-dependent pathways, the RecBCD pathway, and the RecF pathway (Kowalczykowski et al, 1994; Kuzminov, 1999). RecB, RecC and RecD function as a heterotrimeric complex that processes DSBs and is required for recombination during conjugation or transduction (Roman and Kowalczykowski, 1989). Interestingly, the recombination and repair defects in recBC mutants are efficiently suppressed by mutations in sbcA, sbcB, or sbcCD as long as the RecF pathway is functional (Bidnenko et al, 1999). RecF, RecO, and RecR proteins form an epistatic group important for repair of single-stranded (ss) DNA gaps (SSGs) such as when replication is prematurely disrupted (Horii and Clark, 1973; Wang and Smith, 1984; Kolodner et al, 1985; Asai and Kogoma, 1994; Courcelle et al, 1997; Courcelle and Hanawalt, 2003). Homologs of RecFOR genes are found more frequently within bacterial genomes than are homologs of RecBCD (Rocha et al, 2005). For example, RecBC homologs are absent in the extremely DNA damage-resistant bacteria Deinococcus radiodurans, whereas homologs of RecFOR are present (Makarova et al, 2001). Several eukaryotic proteins such as WRN, BLM, RAD52, and BRCA2 share homology or functional similarities to RecF pathway genes, and are associated with a predisposition to cancer and aging when mutated (Karow et al, 2000; Mohaghegh and Hickson, 2001; Kowalczykowski, 2005; Yang et al, 2005). The RecFOR proteins are thought to act by promoting the formation of RecA nucleoprotein filaments on ssDNA, also called presynaptic complexes, by overcoming the inhibitory function of ssDNA-binding protein (SSB) (Brent and Ptashne, 1980; Kolodner et al, 1985; Sassanfar and Roberts, 1990; Umezu et al, 1993; Cox et al, 2000; Courcelle et al, 2001; Courcelle and Hanawalt, 2003; Morimatsu and Kowalczykowski, 2003). The DNA-bound RecA filament is important for rec-dependent repair and serves as a sensor that signals the upregulation of the cellular ‘SOS’ genes in response to DNA damage. This function places RecF, -O and -R in a ubiquitous family of recombinational mediators (RMs) that are found in all organisms (Beernink and Morrical, 1999; Kowalczykowski, 2005). The mechanism by which RMs facilitate loading of RecA-like recombinases on SSB or RPA protected ssDNA is not understood. Among RecF, -O, and -R, RecO is the least conserved. It possesses a DNA strand annealing activity that is similar to that of eukaryotic Rad52 (Luisi-DeLuca and Kolodner, 1994; Kantake et al, 2002; Makharashvili et al, 2004). Both proteins can anneal ssDNA coated by cognate ssDNA-binding proteins, suggesting specific protein–protein interactions between RecO and SSB. The crystal structure of RecO suggests potential sites of interaction with other proteins and DNA (Makharashvili et al, 2004; Leiros et al, 2005). RecR, the most conserved of the three proteins, can interact with either RecO or RecF and has been proposed to functionally tether these proteins together, potentially mediating the sequential steps required for presynaptic complex formation to occur (Umezu and Kolodner, 1994; Webb et al, 1997; Bork et al, 2001; Morimatsu and Kowalczykowski, 2003). Together, RecO and RecR can promote RecA nucleation on SSB-coated ssDNA (Umezu et al, 1993; Shan et al, 1997; Bork et al, 2001). The crystal structure of RecR in D. radiodurans revealed a tetrameric clamp that could encircle dsDNA (Lee et al, 2004). The tetrameric clamp structure is also conserved in crystals of RecR from Haemophilus influenza (data in preparation). A DNA clamp may serve as an important component that tethers RM complexes to sites of DNA damage. The observation that RecR does not bind DNA by itself under physiological conditions (Webb et al, 1995) suggests that an as-yet unknown clamp-loading activity may be required in the RM reaction. Addition of RecF to in vitro reactions was shown to be required for efficient presynaptic complex formation to occur specifically at ss/dsDNA junctions when only a substoichiometric amounts of RecFOR were present relative to SSB and RecA, even under conditions with the excess of SSB (Morimatsu and Kowalczykowski, 2003). This finding confirmed earlier hypothesis that RecF may properly position RecOR on specific DNA sites to initiate presynaptic complex formation (Sandler and Clark, 1994). Interestingly, the efficient loading of the eukaryotic Rad51 recombinase on RPA-coated DNA by the BRCA2 homolog Brh2 was observed to occur at the identical ds/ssDNA junction substrates (Yang et al, 2005). The specific mechanisms by which RecF recognizes gapped DNA substrates, or coordinates interactions with RecO and -R proteins, are not known. RecF, together with RecR, also prevents RecA filaments from extending beyond SSGs (Hegde et al, 1996; Webb et al, 1997). In E. coli, RecR interacts with RecF only when the latter is bound to DNA (Webb et al, 1999). The separate location of RecF, -O, and -R on chromosome (Rocha et al, 2005) and some genetic studies suggested that RecF may have multiple functions, independent of RecO or RecR (Rangarajan et al, 2002; Kidane et al, 2004). In vitro, RecF binds RecX, thus diminishing the negative regulatory effect of RecX during presynaptic complex formation (Drees et al, 2004; Lusetti et al, 2006). The detailed tertiary structure of RecF would be very instrumental to delineate various RecF activities and particularly to study mechanism of the presynaptic complex formation. The amino-acid sequence of RecF contains three conserved motifs characteristic of ATP-binding cassette (ABC) ATPases: Walker A, Walker B, and a ‘signature’ motif. The ABC ATPases comprise a diverse family of proteins whose functions range from membrane transporters to DNA-binding proteins (review in Hopfner and Tainer, 2003). The latter includes DNA mismatch and nucleotide excision repair enzymes (Ban and Yang, 1998; Obmolova et al, 2000; Junop et al, 2001), structural maintenance of chromosome (SMC) proteins like cohesin and condensin (Strunnikov, 1998), and the DSB repair enzyme Rad50 (Hirano et al, 1995). SMCs and Rad50 are characterized by the presence of a long coiled-coil structural domain inserted between N- and C-terminal halves of the globular head domain (Haering et al, 2002). The ABC-type ATPases exhibit ATP-dependent dimerization, in which the signature motif residues interact with ATP bound to the opposite molecule (in trans) (Junop et al, 2001; Smith et al, 2002; Hopfner and Tainer, 2003; Moncalian et al, 2004). RecF lacks a coiled–coil region, but it does exhibit an ATP-dependent DNA binding and a slow DNA-dependent ATP hydrolysis activity, similar to other SMC-like proteins (Madiraju and Clark, 1992; Webb et al, 1995; Hegde et al, 1996). The SMC-like properties of RecF and their role in recombination mediation reaction have not been extensively characterized. However, a previous study demonstrated that the conserved lysine residue of the ATP-binding motif (Walker A) is critical for RecF function, an observation that would be consistent with the idea that ATP hydrolysis is involved (Sandler et al, 1992; Webb et al, 1999). In this report, we describe the high-resolution structure of RecF from D. radiodurans. We present evidence that the three SMC conserved motifs are important for RecF function in vivo, and demonstrate that ATP binding triggers RecF dimerization. The RecF structure is highly homologous to the head domain of Rad50, including α-helices from which the long coiled-coil domain of Rad50 originates, implying a conserved mechanism of DNA binding and recognition of the boundaries of dsDNA regions by both proteins. The structural conservation to Rad50 permits us to model the probable mechanism for binding ds/ssDNA junction by the RecF dimer. Based on the SMC-like dimerization of RecF and together with previously reported ATP- and DNA-dependent interactions with RecR, we propose that RecF dimerization on DNA may serve to place an RecR tetramer clamp on DNA. Results RecF structure RecF was crystallized in a monoclinic space group with one molecule in an asymmetric unit. The structure was solved at a resolution 1.62 Å using native and selenomethionine protein derivative crystals. The final R-factor and free-R were 16.3 and 21.2%, with excellent geometry (Supplementary Table S1). The RecF structure is composed of two domains (Figure 1A). The ATPase domain 1 is formed by two β-sheets wrapped around a central α-helix A and is similar to the Lobe I subdomain of the Rad50 head domain (Figure 1B and C). The first antiparallel β-sheet is formed by six N-terminal β-strands (2↓ 1↑ 4↓ 5↑ 6↓ 7↑). The β3 and β8 form parts of the second β-sheet, which is otherwise composed of the C-terminal β-strands (15↓ 14↑ 3↑ 13↑ 12↑ 8↑). These two β-layers form an almost continuous β-sheet with a small gap between β2 and β15. Domain 2 is mostly α-helical and is similar to the Lobe II subdomain of the Rad50 head domain. The three α-helices, αB, αG, and αH, form a layer on top of the C-terminal β-sheet of the first domain. The helices αC, αD, and an antiparallel 3-stranded β-sheet (9↑ 10↓ 11↑), with insertion of two short helices αE and αF, form the remainder of the second domain. In Rad50, helices corresponding to αC and αD extend into a long coiled-coil region, which is absent in RecF. The nucleotide-binding Walker A motif (P-loop) is at the N-terminus of αA, whereas the Walker B motif is at the C-terminus of β12 and the signature ABC motif resides in the second domain at the beginning of αG. Figure 1.The structure of RecF is similar to the head domain of Rad50. (A) A ribbon representation of RecF in stereo view is shown. β-strands are numbered and shown in yellow and α-helixes are lettered and shown in red. Walker A, B, and signature motifs are highlighted in green and designated by letters A, B, and S correspondingly. Domains I and II are identified on the right. (B) A ribbon representation of the Rad50 (1ii8) structure with same color-coding of secondary structure elements. (C) A ribbon representation of the superimposed ATPase subdomains for RecF (cyan) and Rad50 (orange). (D, E) Superposition of Lobe II subdomains of RecF (cyan) and Lobe II of Rad50 (orange). The superposition was performed by overlaying the whole subdomains, but presented in two separate parts for clarity. Structural superposition and figures in the paper were performed with the program ICM (http://www.molsoft.com)(Cardozo et al, 1995). (F) Stereoview of the superposition of RecF with two different conformations of Rad50 head domain. The Lobe I domain of RecF is shown in dark blue and Lobe II in cyan. Two conformations of the Rad50 head domains are shown: the conformation found in monomeric structure (1ii8) is shown in yellow, whereas conformation of head domain from the dimeric structure (1f2u) is shown in red. The structures were superimposed by ATPase subdomains. ATPγS molecule bound to Rad50 are shown in green stick representation. Download figure Download PowerPoint Structural similarity with Rad50 Almost all structural elements found in RecF are present in Rad50, suggesting that there is a strong evolutionary connection between these proteins. The ATPase domain and the three α-helices above the second β-sheet (αB, αG, and αH) are common to all ABC-type ATPases. The helix αA and the surrounding β-strands of RecF superimpose with their structural counterparts of Rad50 with a root mean square deviation (r.m.s.d.) of 2.7 Å for 107 Cα atoms (Figure 1C). The structural elements surrounding the Walker A motifs of each protein have almost identical conformations. In contrast, β4–β7 on the opposite side of domain 1 forms a more compact structure in RecF than that of Rad50. RecF also lacks short α-helixes inserted instead of β7 and β8. Surprisingly, the second domain of RecF, which is more diverse among ABC ATPases, shares an even higher degree of similarity to Rad50 than the ATP-binding domain. Essentially every α-helix and β-strand of Rad50's Lobe II subdomain is also present in RecF (Figure 1D, and E). Ninety-three Cα atoms of this domain are superimposed with their equivalent part in Rad50 with an r.m.s.d. of 2.2 Å. Likewise, in another SMC protein (PDB id: 1w1w (Haering et al, 2004)), 118 Cα atoms of this same region superimpose with an r.m.s.d. of 2.6 Å. Although the long coiled-coil inserts, characteristic of Rad50 and SMC proteins, are absent in RecF, the RecF's αC and αD helices overlap with the helices of Rad50 that extend into the coiled–coil domain. The only structural addition in this domain, which is unique to RecF, is the extension of the loop between β9 and β10 with two short α-helices (αE and αF). The orientation of the subdomains relative to each other is slightly different between the two proteins (Figure 1F). In RecF, Lobe II is rotated around the Lobe I away from position of Lobe II in Rad50. As a result, the distance between the conserved serine in signature motif of RecF and Rad50 is 18 Å when the structures are superimposed by Walker A and B motifs. The different orientation of Lobe II subdomain in RecF may be a result of the more compact structure of Lobe I subdomain lacking two α-helixes (αB and αC in Rad50) at the C-terminal end of αA. Interestingly, this alignment moves the αH of Lobe I domain together with the Lobe II domain. The direction of this rotation is almost perpendicular to the direction of the Lobe II domain rotation that is observed in Rad50 upon ATP-dependent dimerization (Figure 1F). Structure-based sequence alignment Based on structural superposition, we derived a proper sequence alignment between Rad50 and RecF (Figure 2). Remarkably, most of the conserved residues in both domains of the bacterial RecF protein are also well preserved in Rad50, including the Walker A, Walker B, signature motifs, D-loop, and other residues. The only exception is the Q-loop, but in this case, a structurally similar loop is preserved in RecF. Figure 2.Multiple sequence alignment of 11 RecF proteins and P. furiosus Rad50. Sequence alignment was performed using the ClustalW program and plotted with the ESPript program (Thompson et al, 1994; Gouet et al, 1999). Similar residues as identified by the default ESPript parameters (Risler, global score 0.7) are highlighted in yellow, identical residues are in red. Alignment of Rad50 head domain sequence (i8 in diagram) is based on the structural alignment of RecF and pfRad50, with Rad50 residues highlighted in yellow and red if they are similar or identical to RecF consensus. Secondary structure elements of RecF are shown above the sequences. Conserved motifs are identified by green bars under the sequences with the letters A, B, S, and D for Walker A, B, signature and D-loop motifs. Magenta bars show residues involved in the predicted RecF dimerization (above sequences) and the known dimeric crystal structure of Rad50 (PDB ID: 1f2u) (below sequence). Residues that make up the partially buried charged cluster in Lobe II are identified by blue asterisks under the sequence. Abbreviations for organisms are as follows: DR, D. radiodurans; HI, H. influenza; EC, E. coli; PA, P. aeruginosa; PM, P. multocida; RC, R. conorii; Fs, F. nucleatum; Tp, T. pallidum; BM, B. melitensis; CA, C. acetobutylicum; CT, C. trachomatis; i8, P. furiousus Rad50. Download figure Download PowerPoint A number of additional residues are also conserved between bacterial RecF and eukaryotic Rad50. The R15 between β1 and β2 is involved in the dimerization, nucleotide binding, and interacts with the potential DNA binding regions. The N35 of the P-loop is an essential part of the dimerization interface and is conserved only between RecF and Rad50 proteins (Figure 2; Hopfner et al, 2000), but not in the P-loops of other ABC ATPases. Several polar residues in Lobe II of both proteins are fully or partially buried, surrounding the conserved R190 (Figures 2 and 4D). These residues include R131 (R153), R132 (E154), D136 (R158) of αB (αD), and R190 (R741) of αD (αE), where the corresponding residues and secondary structural elements of Rad50 are shown in parentheses. The preservation of these residues in such an unusual conformation across species and protein families strongly implies that they are important for protein function, possibly allosteric regulation. Figure 3.(A) D. radiodurans RecF is a DNA-dependent ATPase. The phosphate release over time by wild type (solid lines) RecF in the absence (black circles) and presence of DNA substrates: ss (black squares)—30-nt long ssDNA (TAT CCG CAG AGT TGG CTG GTA GTT CAG CCC); ss2 (black diamonds)—15-mer ssDNA (TAT CCG CAG AGT TGG); ds (black inverted triangles)—30-mer ssDNA annealed with a 30-mer complimentary strand; ds/ss (black triangles)—30-mer ssDNA annealed with a 15-mer complimentary strand (15-mer dsDNA with a 15-mer 3′ ssDNA tail). DNA concentration is 17 μM. ATPase activity of RecF mutants in the presence of 30-mer dsDNA is shown by dashed line with gray filled circles for S268R (signature motif) mutant, and by dotted line with gray filled triangles for D300N (Walker B motif) mutant. Lack of ATPase activity by lysine to arginine substitution in Walker A motif was previously reported (Webb et al, 1999). The insert diagram represents the phosphate release dependence on RecF concentration at 30 and 60 min in the presence of dsDNA. (B) Cells expressing RecF proteins mutated in the Walker A, B, or signature motif fail to complement a recF mutant in E. coli UV resistance assay. The survival of wild type (lane 1), recF vector (2), recF expressing the normal RecF protein (3), and recF expressing the site-specific RecF mutations K36R (4), K36M (5), S270R (6), or D303N (7) are shown following exposure to the indicated dose of UV irradiation (note the numbering of residues accordingly to the E. coli sequence). Download figure Download PowerPoint Functional role of SMC conserved motifs To address the role of the conserved motifs in RecF function, site-specific mutations were introduced into E.coli RecF to see if they could functionally complement an E. coli recF mutant. Two independent mutations were introduced into the Walker A motif (K36R and K36M; note that the E. coli sequence numbering is used, and not D. radiodurans). The K36R mutation prevents ATP hydrolysis, but not nucleotide binding. This mutation has been introduced previously in E. coli and is shown to be critical for RecF function (Sandler et al, 1992; Webb et al, 1999). A methionine at position 36 in place of lysine (K36M) has been shown in other Walker A motifs to prevent ATP binding. In the Walker B motif, we changed the aspartic acid at position 303 to an asparagine (D303N). In other SMC proteins, this change traps ATP in its transition state and stabilizes dimerization of the homodimer (Smith et al, 2002). Finally, in the signature motif, we substituted the serine at position 270 to arginine (S270R). In Rad50, the equivalent mutation interferes with ATP-dependent dimerization (Moncalian et al, 2004). The mutations were introduced in the E. coli recF in the pQE-9 vector. This RecF expression plasmid complemented the UV hypersensitivity of a recF mutant, even when expression was not induced by IPTG (Figure 3B), consistent with previous studies that found a low level of RecF expression was sufficient for function in vivo (Sandler and Clark, 1993). Each of the four RecF mutations failed to complement the UV hypersensitivity of the recF mutant, indicating that all three major SMC motifs in RecF are important for its role in UV resistance and supporting the idea that the roles of these conserved motifs are likely to be similar to their counterparts in other SMC proteins. Figure 4.Model of RecF dimer and DNA binding. (A) A ribbon representation of potential RecF dimer with Lobe I and II of one monomer colored in yellow and orange respectively, and those of the second monomer in cyan and dark blue. The bottom panel is the view from the top of the orientation shown in upper panel. The B-form dsDNA is shown as gray sticks. (B) Surface representation of RecF dimer color-coded according to the surface electrostatic potential, calculated with REBEL (Rapid Exact-Boundary Electrostatics) method as implemented in the ICM program with maximum color potential set at ±5 kT/e (Totrov and Abagyan, 1996; Totrov and Abagyan, 2001). Molecules are shown in the same orientations as in (A). DNA is shown in green. (C) The proposed model of asymmetric DNA binding to RecF dimer. ATPase domains are shown as rectangles and Lobe II domains as ovals with same color coding as in (A). ATP-binding sites are depicted as green ovals. DNA is schematically shown on the top in black and gray. Binding of dsDNA (black) in vicinity of one ATP-binding site and ssDNA (gray) to the other is speculated to have different effect on conformational changes and ATPase properties of each half of the dimer as depicted by different filing modes of each subunit. (D) The RecF dimer is shown in worm representation with conserved RecF residues of one monomer shown as sticks and color-coded accordingly to their polarity. Two residues of conserved polar residues buried in Lobe II subdomain are numbered. Download figure Download PowerPoint DNA-dependent ATPase activity of RecF We observed a DNA-dependent ATPase activity of D. radiodurans RecF (Figure 3A) similar to that seen for the E. coli RecF (Webb et al, 1999). The rate of ATP hydrolysis was two-fold higher in the presence of dsDNA (0.22 min−1) than with ssDNA (0.1 min−1). In the presence of the ss/dsDNA junction, the rate was 0.13 min−1 and no ATPase activity was observed without DNA. Importantly, the signature motif mutation (S268R) prevented ATP hydrolysis, similar to that of Walker B D300N mutation (Figure 3A). The ATP-dependent dimerization of RecF The ATP-dependent protein dimerization is a key step in regulating the function of all ABC ATPases that have been characterized to date (for a review, see Hopfner and Tainer, 2003). However, as most ABC ATPases are function as part of larger heterooligomeric complexes, it has been difficult to precisely identify when and what role the dimerization has in a complex multistep reactions. In case of RecF, the dimerization could be important for DNA binding, recognition of ss/dsDNA junction, interactions with other protein partners, such as RecR, or for a combination of these events. As a first step towards dissecting this question, we examined whether RecF oligomerization occurred in the presence of ATP. Initial attempts to utilize the size-exclusion chromatography (SEC) alone were complicated owing to the tendency of RecF to aggregate and due to potential nonspecific interactions of RecF with gel-filtration matrices resulted in late elution of RecF comparatively to standard protein markers (Supplementary Figure S1). Delay of RecF elution varied depending on different buffers, presence of nucleotide, and gel-filtration matrices. Therefore, the molecular weight (MW) of the eluted protein species was directly measured by the subsequent multiangle static light scattering analysis (SLS) (Supplementary Figure S1). Under most conditions, including the presence of 2 mM ATP in a low-salt (0.1 M KCl) running buffer, the SLS measurements resulted in a MW of 38±10% kDa, corresponding to a monomer. Only when highly concentrated protein was loaded (2 mg/ml and higher) on the column, equilibrated with 2 mM ATP in low salt, the SLS estimated MW was 78±10% kDa, even though the elution volume of the dimer was closer to those of 44 kDa marker (BioRad). Thus, RecF forms an ATP-induced dimer even in the absence of DNA, although requirement for high concentration pointed to relatively low dimerization constant. These data also explained previously reported apparent monomeric state of E. coli RecF in the presence of 1mM ATP as estimated by gel filtration method only (Webb et al, 1999). We also addressed the role of SMC conserved motifs in ATP-dependent dimerization of RecF. Because of complications connected with the protein solubility and dimer stability during gel filtration, we utilized the dynamic light scattering (DLS) method, which allows direct measurements of hydrodynamic radius (Rh) of the complexes in solution under equilibrium conditions, as opposed to the SEC method. The observed Rh are shown in Table I. Under conditions that the SEC/SLS analysis demonstrated only the presence of the monomer (no ATP or high salt 1 M KCl), the Rh of RecF was determined to be in a range of 3.4–3.7 nm. In an ideal sphere approximation, it corresponds to an apparent MW of 60–68 kDa, and the difference with the expected MW of RecF of 40 kDa can be explained by the elongated shape of the globule. Under conditions favorable for the dimer formation (see above), the Rh was between 4.2–4.5 nm, corresponding to the almost doubled apparent MW of 100–115 kDa. The standard deviations (polydispersity) of all reported DLS measurements were less than 15%, indicating monodisperse distributions. The differences between mean values were in 2–4% range under similar protein concentration conditions. Interestingly, the stable dimer was observed only with RecF concentrations above 0.2 mg/ml (5 μM) at 20°C, pointing to the low dimerization constant in a micromolar range. It is possible, that other interactions with DNA or RecR can farther stabilize RecF dimer. Table" @default.
• W2115410512 created "2016-06-24" @default.
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• W2115410512 date "2007-01-25" @default.
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• W2115410512 title "Structural conservation of RecF and Rad50: implications for DNA recognition and RecF function" @default.
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• W2115410512 doi "https://doi.org/10.1038/sj.emboj.7601537" @default.
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