Crystal structure and DNA-binding analysis of RecO from Deinococcus radiodurans
2005; Springer Nature; Volume: 24; Issue: 5 Linguagem: Inglês
10.1038/sj.emboj.7600582
ISSN1460-2075
AutoresIngar Leiros, Joanna Timmins, David R. Hall, Seán McSweeney,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle17 February 2005free access Crystal structure and DNA-binding analysis of RecO from Deinococcus radiodurans Ingar Leiros Ingar Leiros Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Joanna Timmins Joanna Timmins Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author David R Hall David R Hall Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Sean McSweeney Corresponding Author Sean McSweeney Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Ingar Leiros Ingar Leiros Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Joanna Timmins Joanna Timmins Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author David R Hall David R Hall Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Sean McSweeney Corresponding Author Sean McSweeney Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France Search for more papers by this author Author Information Ingar Leiros1,‡, Joanna Timmins1,‡, David R Hall1 and Sean McSweeney 1 1Macromolecular Crystallography Group, European Synchrotron Radiation Facility, Grenoble, France ‡These authors contributed equally to this work *Corresponding author. Macromolecular Crystallography Group, European Synchrotron Radiation Facility, BP 220, 6 rue Jules Horowitz, 38043 Grenoble Cedex 9, France. Tel.: +33 4 76 88 23 62; Fax: +33 4 76 88 21 60; E-mail: [email protected] The EMBO Journal (2005)24:906-918https://doi.org/10.1038/sj.emboj.7600582 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The RecFOR pathway has been shown to be essential for DNA repair through the process of homologous recombination in bacteria and, recently, to be important in the recovery of stalled replication forks following UV irradiation. RecO, along with RecR, RecF, RecQ and RecJ, is a principal actor in this fundamental DNA repair pathway. Here we present the three-dimensional structure of a member of the RecO family. The crystal structure of Deinococcus radiodurans RecO (drRecO) reveals possible binding sites for DNA and for the RecO-binding proteins within its three discrete structural regions: an N-terminal oligonucleotide/oligosaccharide-binding domain, a helical bundle and a zinc-finger motif. Furthermore, drRecO was found to form a stable complex with RecR and to bind both single- and double-stranded DNA. Mutational analysis confirmed the existence of multiple DNA-binding sites within the protein. Introduction Maintaining genomic integrity is crucial in all organisms. DNA damage can be caused by many factors, for example, ultraviolet (UV) radiation, γ-radiation, chemical mutagens and even intrinsic errors during DNA replication. Deinococcus radiodurans tolerates ionising radiation at doses lethal to other organisms and is capable of surviving 5–30 kGy of ionising radiation (Minton, 1994), whereas most other organisms cannot survive doses greater than 50 Gy. Such massive radiation doses are estimated to induce several hundred double-strand breaks, thousands of single-strand gaps and about 1000 sites of DNA base damage per chromosome (Battista, 1997 and references therein). The two RecA-dependent DNA repair pathways (RecBCD and RecFOR), which normally operate independently of each other, are important in double-strand break repair and post-replication daughter-strand gap repair, respectively (Morimatsu and Kowalczykowski, 2003). The primary function of these two pathways in bacteria is the recombination-mediated repair of stalled or collapsed DNA replication forks (Cox, 2001). In D. radiodurans, RecD is the only protein in the RecBCD pathway that has so far been identified, whereas all members of the RecFOR pathway have been identified and annotated (Makarova et al, 2001). Consequently, it has been speculated that the RecFOR pathway might replace some of the RecBCD pathway function in D. radiodurans (Aono et al, 2003). It has also been shown that members of the RecFOR family of proteins can recover the viability of certain RecBCD mutants in Escherichia coli (Amundsen and Smith, 2003). The RecFOR pathway comprises several proteins, for example, RecQ (3′-5′ helicase), RecJ (5′-3′ nuclease), and the RecF, RecO and RecR proteins, where the latter three, perhaps in a molecular complex, displace single-stranded DNA (ssDNA)-binding protein (SSB) and facilitate the production of a RecA-coated ssDNA filament (Amundsen and Smith, 2003). The RecFOR proteins have been found to be important in protecting the nascent lagging strand when replication forks are stalled on UV radiation damage sites (Chow and Courcelle, 2004). Mutants in any of the RecFOR proteins cause E. coli to become both hypersensitive to UV radiation and show extensive degradation in the nascent lagging strand. This degradation, in turn, is limited in RecF, RecO or RecR mutants, when in addition either RecJ or RecQ is inactivated (Chow and Courcelle, 2004). It has been shown that purified RecF, RecO and RecR proteins can form a complex in an apparent equimolar ratio (Umezu et al, 1993; Hegde et al, 1996), and that the E. coli RecO protein interacts physically with RecF, RecR and SSB (Umezu and Kolodner, 1994; Hegde et al, 1996). The E. coli RecO protein has also been found to have an ability to bind SSB-coated ssDNA and to anneal complementary ssDNA strands regardless of whether the DNA was preincubated with SSB or not (Kantake et al, 2002). RecR from D. radiodurans (drRecR) has been found to form tetramers in a ring-like structure (Lee et al, 2004). A central hole in the tetramer has a diameter of 30 Å and is suited for binding double-stranded DNA (dsDNA). In the crystal structure of drRecR, two such tetramers are interlocked and it is therefore believed that RecR tetramers can open and close in order to bind DNA (Lee et al, 2004). The RecFOR complex is specific for the dsDNA–ssDNA junction in the lagging strand of DNA at a stalled replication fork. Mapping this specificity onto the individual components of the RecFOR complex is crucial in understanding the ongoing processes at such sites. Obtaining biochemical and structural information about the involvement of RecO in DNA binding and its interactions with other DNA repair proteins is important. As a first step in understanding the structural aspects of its involvement in DNA repair, the crystal structure of D. radiodurans RecO (drRecO) has been determined. Mutational analysis indicates the regions of the protein that are involved in binding DNA, and reveals that there are multiple sites interacting with DNA, one of which appears to be a species-specific site. The crystal structure and mutational analysis of drRecO, in addition to the recently published structure of drRecR, serve as starting points for comprehension of DNA repair not only in D. radiodurans but also in a wider context. Results The structure of RecO from D. radiodurans The crystal structure of drRecO was determined to 2.4 Å resolution by the single-wavelength anomalous dispersion (SAD) method. drRecO has dimensions of approximately 60 × 30 × 30 Å3 and is composed of an N-terminal oligonucleotide/oligosaccharide-binding (OB) fold region (Murzin, 1993), a three-helix bundle, a Cys4 zinc-finger motif and finally a group of four helices inserted between the three-helix bundle and the zinc-finger motif (Figure 1). There are two molecules per crystallographic asymmetric unit (a.s.u.) with a root-mean-square (r.m.s.) deviation between the two monomers of 0.68 Å for 220 aligned Cα atoms. The buried surface area for each monomer is on average 7.1% (825 Å2), suggesting that the two molecules per a.s.u. is a crystal packing effect. This is in agreement with a single peak at a size consistent with a monomer of drRecO in size exclusion chromatography and in accordance with chemical crosslinking experiments (data not shown). The N-terminal OB fold domain in drRecO consists of residues 1–79 in five highly curved β-strands (β1–β5; Figures 1 and 2), which form a β-barrel. There is normally a capping helix (or occasionally a coil) between β3 and β4 in the classic OB fold (Bochkarev and Bochkareva, 2004), which seals one end of the β-barrel. In drRecO, these residues are flexible and absent from electron density in both monomers (residues 41–45). The OB fold domain is followed by one turn of α-helix (α1) and three long α-helices (residues 86–144; α2–α4) stacked tightly into an antiparallel α-helical bundle. The helical bundle is followed by an unusual Cys4 zinc-finger motif (residues 153–176) with the sequence CX2CX16CX2C. A zinc atom, tetrahedrally coordinated by Cys153, Cys156, Cys173 and Cys176, is well defined in electron density in each monomer. Four additional helices (residues 184–239; α5–α8) are spatially inserted between the zinc-finger and the helical bundle. Figure 1.Topology sketch (A) and ribbon illustrations (B, C) of the secondary structure elements in drRecO. α-Helices are shown in red and β-strands in yellow. The zinc atom in the zinc-finger motif is illustrated as a cyan sphere. The illustration given in (C) is rotated 90° from the view in (B). Download figure Download PowerPoint Figure 2.Sequence alignments of RecO from D. radiodurans (TrEMBL, Q9RW50), T. thermophilus (TrEMBL, Q72L07), E. coli (Swiss-Prot, P15027), Magnetospirillum magnetotacticum (NCBI, ZP_00048866), Thermobifida fusca (NCBI, ZP_00058304), Streptomyces coelicolor (Swiss-Prot, Q9L2H3), Corynebacterium efficiens (TrEMBL, Q8FNG1) and Tropheryma whipplei (TrEMBL, Q83MZ1). Secondary structure elements from the crystal structure of drRecO are indicated with spirals (α-helices) and arrows (β-strands) above each row of the sequence alignment. Amino-acid residues having water-accessible surfaces greater than 10 Å2 are indicated with 'E' for external, otherwise indicated with 'I' for internal. The four cysteine residues in the zinc-finger motif of drRecO are indicated with red triangles. Equivalent residues conserved in more than 60% (5) of the sequences are boxed and identical residues are shown on a red background. Download figure Download PowerPoint Structural comparison to other proteins drRecO has 25% sequence identity to Thermus thermophilus RecO and 17% to E. coli RecO. Sequence alignments of bacterial RecO proteins show that the conservation of the N-terminal part of the proteins is higher than for the C-terminus (Figure 2). Using only the OB fold domain as a search template, DALI (Holm and Sander, 1993) gave good scores for several nucleotide-binding proteins, with the best match being human replication protein A (RPA; Bochkarev et al, 1997), E. coli aspartyl-tRNA synthetase (Eiler et al, 1999) and SSB proteins from E. coli (Raghunathan et al, 2000) and Sulfolobus solfataricus (Kerr et al, 2003), followed by the breast cancer susceptibility gene 2 encoded protein (BRCA2), which also aligns well to the OB fold domain of drRecO. RPA is the human equivalent of SSB and is a heterotrimer consisting of a total of six OB fold domains, of which four interact with ssDNA (Bochkareva et al, 2002 and references therein). A structural alignment where the DNA-bound structures of RPA and SSB are superimposed onto the OB fold of drRecO displays some similarities in the DNA-binding region. Within the OB fold, ssDNA binding is generally mediated by three distinct structural elements: the β1–β2 loop, the β4–β5 loop and the C-terminal part of β3, where the first provides positively charged residues to form ion-pair(s) with the ssDNA phosphodiester backbone and the latter two each provide one conserved aromatic moiety (Bochkarev and Bochkareva, 2004). These residues are illustrated in Figure 3, where it can be seen that, except for the β4–β5 loop, which is shorter in drRecO than in the other OB-loop-containing proteins, positively charged residues in drRecO occupy similar positions. In all the compared SSB proteins, there are at least two aromatic residues that have been shown to be important in ssDNA binding. In drRecO, no such aromatic residues can be found and there could be a tendency for substitution of aromatic residues in RPA/SSB into positively charged residues in drRecO. Among RecO proteins (Figure 2), R16 and K35 in drRecO are conserved in being positively charged, thus further strengthening this possibility. Figure 3.Structural comparison between some aligned OB fold domains. (A) drRecO, (B) S. solfataricus SSB (1O7I), (C) E. coli SSB (1EYG) and (D) human RPA70 (1JMC). All the displayed OB folds are coloured in rainbow colours ranging from blue at the N-terminus to red at the C-terminus. The nucleotide-interacting residues are generally found in the cleft on the right-hand side of the displayed domain. Residues in the β1–β2 loop and in the β4–β5 loop line this cleft. The OB fold domain in drRecO is shorter (81 residues including six missing residues in the β3–β4 loop) than any of the other structurally aligned proteins (85–108). Some of the semiconserved residues thought to be important in ssDNA binding are shown as sticks. In (C, D), the cocrystallised ssDNA is shown as brown coils. (E) Semiconserved residues between the four proteins, also illustrated in (A–D). N/C, not conserved. Download figure Download PowerPoint Zinc-fingers have long been considered as important mediators of protein–DNA interactions (Branden and Tooze, 1991). Typically, they have the secondary structure succession β–β–α where the α-helix contacts DNA through major-groove interactions. In drRecO, the α-helix is not formed (Figure 4A). A few zinc-finger motifs with resemblance to drRecO were manually identified and aligned, among them being the zinc-finger from the crystal structures of drRecR (Lee et al, 2004) and the Sec23/24 heterodimer (Bi et al, 2002), where additional residues around the zinc coordination sphere (mainly the β–β segment) could be structurally aligned with the drRecO zinc-finger motif (conserved β-strands shown in dark blue in Figure 4A–C). The zinc-finger motif in the RecFOR family member RecQ from E. coli (Bernstein et al, 2003) lacks conservation of the β–β segment and is structurally dissimilar to RecO (Figure 4D). However, the importance of the zinc-finger motif in drRecO is questionable as the E. coli and T. thermophilus RecO proteins both lack the C-terminal zinc-finger motif found in many bacterial RecO proteins. Figure 4.Comparison of Cys4 zinc-finger motifs. (A) Stereo presentation of the zinc-finger in drRecO. The four cysteine residues coordinating the zinc atom are illustrated in a ball-and-stick presentation and the zinc atom is shown as a cyan sphere. The electron density maps are 2mFo−DFC maps contoured at 2σ (blue) and 7σ (red), respectively. The cysteine residues and the zinc atom were all omitted from the refinement. The secondary structure elements are labelled as in Figure 2. (B–D) Zinc-finger motifs from Sec23 (1M2V), RecR (1VDD) and RecQ (1OYW). Secondary structure elements are numbered relative to the figures. β-Strands in dark blue are conserved. Download figure Download PowerPoint Although the two most striking structural features of drRecO are the OB fold domain and the zinc-finger motif, the presence of the three-helix bundle and the C-terminal helical inserts between the OB fold domain and the zinc-finger should not be neglected. In particular, a patch in the α5–α6 region (190-RHAVRRTVR-200), which is not conserved among the aligned RecO proteins, is rich in positively charged residues, all of which are exposed to solvent and could be of importance in a species-specific function of drRecO (discussed later). The RecOR complex E. coli RecR is a dimer in solution and interacts with RecO (Umezu and Kolodner, 1994; Hegde et al, 1996). In E. coli, the two proteins are suggested to form a complex in a 1:1 molar ratio, presumably in the form of a 2:2 heterotetramer (Umezu and Kolodner, 1994). They have also been shown to be required in a 1:1 molar ratio for RecA to overcome inhibition by SSB (Umezu et al, 1993). drRecR has been shown to be a tetramer or an octamer in solution in a concentration-dependent manner (Lee et al, 2004). To investigate the nature of the RecOR complex in D. radiodurans, the proteins were expressed individually and copurified in a three-step purification process. As both proteins were tagged with an N-terminal histidine tag, multiple purification steps were used to ensure that a molecular complex was formed. On a final size exclusion chromatography step, the RecOR complex eluted as a single peak (elution volume: 12.1 ml) corresponding to a molecular weight of approximately 150–160 kDa (Figure 5). RecO and RecR (tetramer) alone eluted later from the size exclusion column at 17.1 and 13.5 ml, respectively, agreeing with their calculated molecular weights (26 and 95 kDa). Although monomeric drRecR (23.7 kDa) is smaller than drRecO (26.3 kDa), it migrates as being slightly larger than drRecO on an SDS–PAGE denaturing gel (Figure 5). The ratio of drRecR/drRecO in the RecOR complex was estimated by comparison with known amounts of individually purified drRecR and drRecO on SDS–PAGE, and was found to be in a 2:1 ratio, probably in the form of a 4:2 heterohexamer. Figure 5.Chromatogram of the gel filtration step for the RecOR complex using a flow rate of 0.5 ml/min. The green and violet lines show the absorbance at 280 and 260 nm, respectively. The inset is the SDS–PAGE denaturing gel of the fractions as shown above the chromatogram. Molecular weight markers (M) are shown in kDa. drRecR (23.7 kDa) migrates as being slightly larger than drRecO (26.3 kDa) and the proteins are at an apparent 2:1 ratio in the RecOR complex. Download figure Download PowerPoint DNA binding of recombinant drRecO The DNA-binding properties of drRecO were investigated. Because of the high pI of drRecO, electrophoretic mobility shift assays (EMSA) were analysed by agarose gel electrophoresis in order to visualise migration towards both anode and cathode. The commonly used nondenaturing polyacrylamide bandshift assay did not allow the visualisation of both protein alone and protein–DNA complexes on the same gel within a pH range of 6.5–8.5. Two different approaches were thus used to study the DNA-binding properties of drRecO: (i) visualisation of a DNA bandshift using ethidium bromide staining and (ii) visualisation of a protein bandshift using Coomassie staining. For the first approach, drRecO was incubated with supercoiled plasmid DNA (pcDNA3.1) and the reaction mixtures were analysed by agarose gel electrophoresis (Figure 6A). The addition of drRecO to pcDNA3.1 leads to a dramatic shift of the DNA (Figure 6A, lanes 3 and 4). The addition of Mg2+ to the DNA in the absence of drRecO has no effect on migration (Figure 6A, lane 2). The presence of Mg2+ in lane 4 appears to enhance the binding of drRecO to plasmid DNA, since in its absence a smear of retarded DNA (Figure 6A, lane 3) was observed, as opposed to the well-defined band only just entering the gel when Mg2+ is added to the incubation buffer. This band clearly corresponds to a protein–DNA complex, since in a duplicated experiment when Coomassie stained for protein, a band was observed at the corresponding position (Figure 6B, lane 8). Moreover, the addition of increasing amounts of drRecO to pcDNA3.1 in the presence of Mg2+ resulted in a concentration-dependent shift of the DNA. The incubation of bovine serum albumin (BSA) with pcDNA3.1 did not shift the DNA (Figure 6A, lanes 9 and 10). The shifting of the supercoiled DNA into the well (Figure 6A, lanes 4 and 7) appears to be RecO-specific, while the appearance of a minor form of relaxed circular or linear plasmid DNA may be a result of a nonspecific interaction of protein with supercoiled DNA, since this band is also visible upon incubation of supercoiled DNA with BSA in the presence of Mg2+. To further investigate the DNA-binding ability of drRecO, a second approach was used in which EMSA was carried out at two different pH values (6.8 and 8.3) and analysed by Coomassie staining of the protein. Since drRecO has a high pI (9.2), at a pH of 8.3 with an overall slightly positive charge, the native protein migrates towards the cathode, whereas upon binding to DNA, the overall charge becomes negative and as a result the drRecO—DNA complexes migrate in the opposite direction, that is, towards the anode. At pH 6.8, however, the overall charge of drRecO is significantly more positive and the binding to DNA gives a gel retardation, rather than an inverted migration pattern, caused by a reduction in the overall positive charge of the complex relative to the protein alone. Clear protein bandshifts were observed at pH 8.3 and 6.8 upon incubation of drRecO with ssDNA and dsDNA oligonucleotides (Figure 6B, lanes 2, 3, 10 and 11). However, unlike in the case with supercoiled plasmid DNA, the addition of 40 mM Mg2+ to the reaction mixtures had no significant effect on DNA binding (Figure 6C, lanes 3 and 8). Similarly, the addition of Mg2+ to the protein alone did not alter its migration (Figure 6B, lane 5). In order to further characterise the nature of RecO–DNA interactions, the effect of adding either a metal chelator such as 50 mM Na-EDTA pH 8.0 (to disrupt the C-terminal zinc-finger) or 1 M NaCl (to interfere with ionic interactions) to the protein–DNA mixtures was studied. The analysis of drRecO by size exclusion chromatography in the presence of either 50 mM Na-EDTA pH 8.0 or 1 M NaCl revealed that the overall conformation of drRecO was maintained, as the elution volume of the protein was comparable to that in native conditions (data not shown). Moreover, the addition of either 50 mM Na-EDTA pH 8.0 or 1 M NaCl did not affect the migration of drRecO on the agarose gel (Figure 6C, lanes 11 and 12). The presence of EDTA in the incubation buffer markedly interferes with the binding of drRecO to ssDNA and dsDNA, resulting in a significantly reduced bandshift (Figure 6C, lanes 4 and 9). The resulting protein–DNA complexes are partially retained in the wells and display a more smeared pattern, suggesting that drRecO–DNA interactions are less stable when EDTA is added. A similar smearing is observed upon addition of a high level of salt (1 M NaCl) to the protein–DNA mixtures, although to a lesser extent (Figure 6C, lanes 5 and 10). Figure 6.EMSA of drRecO. (A) Agarose gel electrophoresis staining with ethidium bromide for visualising DNA. Lane M, DNA size markers in base pairs; lane 1, 0.2 μg supercoiled plasmid DNA (pcDNA3.1); lane 2, pcDNA3.1+40 mM MgCl2; lane 3, pcDNA3.1+3 μg drRecO; lane 4, pcDNA3.1+drRecO+40 mM MgCl2; lanes 5–7, as for lane 4 but using 0.05, 0.5 and 2 μg drRecO, respectively; lane 8, as for lane 2; lane 9, pcDNA3.1+5 μg BSA; lane 10, pcDNA3.1+BSA+40 mM MgCl2. (B, C) Agarose gel electrophoresis, but Coomassie staining for protein. (B) Lanes 1–8 at pH 8.3. Lane 1, 5 μg drRecO; lane 2, drRecO+1 μg ssDNA; lane 3, drRecO+1 μg dsDNA; lane 4, drRecO+0.2 μg pcDNA3.1; lanes 5–8, as for lanes 1–4 but with 40 mM MgCl2 added; lanes 6–9, as for lanes 1–4, but at pH 6.8. (C) Lane 1, 5 μg drRecO; lane 2, drRecO+1 μg ssDNA; lane 3, drRecO+ssDNA+40 mM MgCl2; lane 4, drRecO+ssDNA+50 mM EDTA; lane 5, drRecO+ssDNA+1 M NaCl; lane 6, drRecO; lane 7, drRecO+1 μg dsDNA; lane 8, drRecO+dsDNA+40 mM MgCl2; lane 9, drRecO+dsDNA+50 mM EDTA; lane 10, drRecO+dsDNA+1 M NaCl; lane 11, drRecO+50 mM EDTA; lane 12, drRecO+1 M NaCl. Download figure Download PowerPoint DNA binding of drRecO mutants ssDNA and dsDNA were manually docked onto a molecular surface representation of drRecO (not shown). These models were used as a starting point for generating mutants of drRecO. Both single and multiple point mutants were designed to disrupt positively charged residues (mutations into Ala and/or Glu; see Figure 7A). Of the 16 drRecO mutants obtained, all were expressed, and 12 were soluble and successfully purified (Figure 7C). Based on limited proteolysis, all 12 purified mutants appeared to be folded correctly as they had similar degradation patterns to the wild-type protein (Figure 7D). The four insoluble mutants were K35E, R191A/H192A/R195A/R196A, R14A/R16A/R191A/H192A/R195A/R196A and R14A/R16A/K35A/R39A/R191A/H192A/R195A/R196A. In order to investigate the DNA-binding abilities of the drRecO mutants, they were incubated with plasmid DNA as described previously for wild-type drRecO (Figure 6A), and the protein–DNA mixtures were analysed on agarose gels (Figure 7A and B). The mutants could be grouped into three major classes: (1) unaffected DNA binding, (2) partially disrupted DNA-binding capability and (3) loss of DNA binding (as summarised in Figure 8F). Upon binding to the plasmid DNA, wild-type drRecO and DNA are retained in the well (Figure 7A and B, lane 2). This is also the case for mutants K35A and the double-mutant R191A/H192A (lanes 5 and 11), suggesting that they have an unaffected binding to DNA. Similarly, mutant R14A/R16A is only very weakly affected by the mutation. On the other hand, for mutant R16E, and the multiple mutants K35E/R39E and K35E/R195E/R196E, DNA binding appears to be significantly impaired, since DNA and protein migrate in opposite directions (lanes 4, 6 and 14). The remaining six mutants display some residual DNA binding, as can be deduced from the smearing of both the DNA and the protein in Figure 7A and B (lanes 7–10, 12 and 13). Figure 7.Characterisation of drRecO mutants. (A) Agarose gel electrophoresis staining with SYBR safe for visualising DNA. Lanes M, DNA size markers in base pairs; lane 1, 0.2 μg supercoiled plasmid DNA (pcDNA3.1); lane 2, pcDNA 3.1+40 mM MgCl2+5 μg drRecO; lanes 3–14, as for lane 2, but using 5 μg of the mutant indicated above the lanes; lane 15, as for lane 2, but using 5 μg BSA. (B) The gel from (A) but Coomassie staining for protein. (C) 15% SDS-PAGE gel of the wild-type and mutated drRecO used. Lane 1, broad-range molecular marker in kDa; lane 2, 3 μg wild-type drRecO; lanes 3–14, 3 μg of each of the mutants indicated above the lanes in (A); lane 15, 3 μg BSA. (D) Limited proteolysis of wild-type and mutated drRecO. Download figure Download PowerPoint Figure 8.Models for dsDNA interacting with drRecO based on the DNA-binding studies and mutational analysis. In (A), the secondary structure succession is outlined in colours ranging from blue to red. Some positively charged residues are shown for comparison to positive regions seen in the estimated electrostatic surface potentials. Residues mutated in this study are labelled in red. The electrostatic surface potentials in (B–E) are contoured at ±3 kT/e, where red describes a negative and blue a positive potential. dsDNA interacting with drRecO is modelled as sticks in (B, C). Two alternative binding sites involving the OB barrel (bottom) and a positive patch (190-RHAVRRTVR-200) unique for drRecO ending at the zinc-finger (top) are shown. (D) Close-up of dsDNA modelled to interact with the positive patch unique to drRecO with positively charged residues labelled. (E) Close-up of the region in the OB barrel found to be important for dsDNA binding in drRecO. (F) Indication of how well the mutants of drRecO bind to DNA; +++, unaffected DNA-binding ability; +, reduced DNA-binding ability; −, loss of DNA-binding ability. Download figure Download PowerPoint Discussion drRecO is a multidomain protein. The OB fold and helical bundle are the most conserved regions among RecO proteins, suggesting that they may be responsible for carrying out vital and conserved functions shared between RecO proteins. While the three-helix bundle is well conserved, no clear function has been assigned to this domain. The tight packing of this helical bundle suggests that it may play a structural role, maintaining the OB fold and the zinc-finger motif in their respective orientations. This domain organisation has also been shown for the trimerisation core of human RPA, in which one helix is contributed from each subunit (Bochkareva et al, 2002 and references therein). The OB fold domain contributed by RPA14 and the zinc-finger motif in the C-terminal domain of RPA70 are at either ends of the protein complex with a three-helix bundle in the middle. The zinc-finger motif and the inserted helices are more weakly conserved within RecO proteins and may perform species-specific roles. Although RecO consists of well-characterised domains, assigning the exact function to each of these regions is not straightforward. Through purification of the RecOR complex, we have shown that drRecO is able to form a stable protein–protein complex. In addition, by EMSA and mutational analysis, we have both identified the ability of drRecO to interact with DNA and further indicated which regions in drRecO are important in protein–DNA interactions. Residues essential for drRecO to interact with dsDNA have been identified. Protein–protein interactions We have shown that drRecO and drRecR interact to form a tightly associated, stable complex. The size of the RecOR complex was estimated
Referência(s)