Plant Mol Biol (2011) 76:463–471 DOI 10.1007/s11103-011-9736-6
Identification of a soybean chloroplast DNA replication originbinding protein Matthew G. Lassen • Sunita Kocchar Brent L. Nielsen
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Received: 30 June 2010 / Accepted: 10 January 2011 / Published online: 25 January 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS–PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondriallocalized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility M. G. Lassen S. Kocchar B. L. Nielsen (&) Department of Microbiology and Molecular Biology, Brigham Young University, 775 WIDB, Provo, UT 84602, USA e-mail:
[email protected] M. G. Lassen Center for Advanced Drug Research, SRI International, Harrisonburg, VI 22802, USA S. Kocchar National Botanical Research Institute, Lucknow 226001, India
shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication. Keywords Chloroplast DNA replication Origin-binding protein DNA affinity chromatography Single-stranded DNA binding motif
Chloroplast DNA (ctDNA) in higher plants ranges in size from about 120–160 kbp (Kolodner and Tewari 1975), and encodes only some of the genes required for photosynthesis and other chloroplast functions. The remainder of the genes required for chloroplast function is nuclear-encoded, including all genes for ctDNA replication/maintenance and one of the two major RNA polymerases involved in chloroplast gene transcription (Swiatecka-Hagenbruch et al. 2008). CtDNA copy number per cell varies widely, with very high copy number during stages of rapid plant growth and expansion, and very little DNA per cell in mature leaf tissue (Oldenburg and Bendich 2004). Kolodner and Tewari (1975) proposed a double D-loop mechanism for replication of ctDNA, which involves two strand-specific replication origins (ori) that each initiate unidirectional DNA synthesis until they fuse, forming bidirectional replication forks. These two origins are relatively close to each other (*6kbp apart), and their locations in the chloroplast genome have been mapped to the same regions near the
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ctDNA rRNA genes in pea, tobacco and some other species (Kunnimalaiyaan and Nielsen 1997b). A similar double D-loop mechanism has been characterized for ctDNA replication in Chlamydomonas (Wu et al. 1993). Kolodner and Tewari (1975) also reported evidence for rolling circle replication upon completion of replication of a ctDNA molecule by the D-loop mechanism. This suggests that, similar to some phage systems, ctDNA may replicate by more than one mechanism (Nielsen et al. 2010). This is supported by studies from the Koop laboratory that show that targeted inactivation of either or both of the D-loop replication origins was not lethal, although some of the mutants generated led to reduced plant growth rate and reduced ctDNA copy number per cell (Muhlbauer et al. 2002; Scharff and Koop 2007). These reports suggest that D-loop replication may play a role in only certain stages of plant growth, such as maintenance of ctDNA in mature cells. The early rapid expansion of ctDNA may be facilitated by rolling circle or recombination-dependent DNA replication (Nielsen et al. 2010; Scharff and Koop 2006; Oldenburg and Bendich 2003, 2004). Rolling circle replication has also been observed in plant mitochondria (Backert et al. 1996). Initiation of DNA synthesis generally involves specific interactions between an initiator (a trans-acting protein) and a replicator (a cis-acting DNA element) (Jacob, Brenner and Cuzin 1963). Specific interactions between the initiator and the replicator control the initiation of DNA replication. Identification of initiators and replicators has been accomplished in many prokaryotic and eukaryotic organisms. Although the prokaryotic and eukaryotic processes differ in complexity and number of factors involved, initiation is achieved by the same basic mechanism. Initiator protein binding to the ori sequence leads to the initial unwinding of the ori region to prepare for new DNA synthesis. The replication initiation protein may be a single protein (such as bacterial DnaA, RepA) or a complex of protein subunits that together initiate replication (e.g. eukaryotic nuclear ORC1-6), with most containing an ATPase domain, supporting the requirement for ATP hydrolysis in unwinding the ori region (Giraldo 2003). Following unwinding, additional replication proteins involved in initiation are recruited to the ori region. In bacteria, these proteins include a helicase (DnaB) and a primase (DnaG) that together form the primosome. Priming of the DNA template allows DNA polymerase to associate at the unwound ori and proceed with elongation of both the leading and lagging strands. In eukaryotic nuclei, the process is much more complex and involves the coordinate action of cell cycle regulators interacting with multiple initiation complexes at multiple oris (for reviews see Lei and Tye 2001; Bryant et al. 2001).
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Origin-binding proteins (OBPs) have been identified for a variety of replication systems. Bacteria contain DnaA homoloques that function as the initiation protein for chromosomal DNA replication, while many bacterial plasmids require Replication factor A (RepA) in addition to DnaA to control initiation (Messer et al. 1999). In eukaryotic nuclei, an origin recognition complex (ORC) composed of 6 subunits (ORC1-6) binds to multiple replication origins to initiate replication cooperatively with cell cycle regulators (Bryant et al. 2001). There is no significant sequence similarity between bacterial and eukaryotic OBPs, but there are similarities in the types of domains they contain (Giraldo 2003). The only previous sequence-specific ctDNA origin binding protein that has been identified is from Chlamydomonas (Wu et al. 1989). Two replication oris (oriA and oriB) have been identified in chloroplasts of higher plants (Lu et al. 1996; Kunnimalaiyaan and Nielsen 1997a, b). We report here the purification and characterization of an ori-binding protein from total soybean chloroplast protein extracts via sequence-specific DNA affinity chromatography and MALDI-ToF mass spectrometry.
Materials and methods Total chloroplast protein isolation All centrifugation steps were performed at 4°C without braking. Leaf tissue (200–250 g) was harvested from 8 to 9 day old soybean plants and homogenized in STM buffer (0.5 M sucrose, 50 mM Tris–Cl, pH 8.0, 5 mM MgCl2, 5 mM b-mercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride) in a Waring blender with three 5 s low speed bursts and two 3 s high speed bursts. The homogenate was filtered through 4 layers of cheesecloth and 3 layers of miracloth and centrifuged at 1,000 9 g for 10 min. Chloroplast pellets were resuspended in STM buffer and combined with 10 mL of STM buffer/Triton X-100 (7.75 mL STM buffer and 2.25 mL Triton X-100). The volume was then adjusted to 50 mL with STM buffer. Chloroplasts were incubated on ice for 30 min, mixing gently 3–4 times during incubation. Lysed chloroplasts were centrifuged at 6,000 9 g for 20 min. The pellets were discarded and the supernatant was stored on ice. Labelling of 144 bp oriA PCR product A 144 bp oriA region was amplified by polymerase chain reaction. Each 50 ll reaction tube contained 0.5 lM of each primer (CGGGTGAGATCCAATGTAGAT (OA1F) and GCTAAACCTGTGCTCGAGAGAT (OA144R)), 0.2 mM dNTPs, 1.5 mM MgCl2, 1X PCR reaction buffer, 2.5 units of
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Taq polymerase (Invitrogen), and 1 ll DNA template. All PCR reactions were performed in a PTC-150 MiniCycler (MJ Research) under the following conditions: 95°C for 3 min, 30 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min, followed by 72°C for 7 min. A 5 ll aliquot of each reaction tube was analyzed by gel electrophoresis. OriA PCR products from the remainder of each reaction were purified using the QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions. The purified oriA fragment was 3’end-labelled with biotin using the Biotin 3’end DNA Labeling Kit (Pierce) according to the manufacturer’s instructions, and stored at –20°C. Some ctDNA ori fragments (Lugo et al. 2004, see legend to Fig. 1) were radioactively labeled (Sambrook and Russell 2001) for use as probes of a southwestern blot of total chloroplast proteins. Southwestern blot analysis Total proteins from lysed chloroplasts were separated by SDS–PAGE electrophoresis and transferred to a nitrocellulose membrane (MSI; Sambrook and Russell 2001). After transfer, the proteins on the blot are renatured by incubating the blot in binding buffer (50 mM Tris pH 8.0, 1 mM dithiothreitol, 0.3% Tween 20, 150 mM NaCl). Radioactively labeled probes from different clones of either minimal ctDNA ori region (Lugo et al. 2004; see Figure 1 legend for details) or from pUC19 lacking an insert as a control were mixed with an excess of nonspecific poly (dIdC) competitor (10 lg/mL) in binding buffer and incubated with strips of individual lanes of the gel overnight at room temperature. The membrane strips were then washed three times (10 min each) with binding buffer, wrapped in clear plastic wrap and exposed to x-ray film.
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Preparation of DNA affinity column Preparation of oriA-coupled CNBr-activated Sepharose was performed according to Ausubel et al. (1993) with slight modifications. Three grams of CNBr-activated Sepharose 4B powder (Amersham Biosciences) was placed into a 15-mL polypropylene tube and hydrated with 10 mL of 1 mM HCl, mixing gently. The slurry was transfered to a ceramic funnel with a glass fiber filter and washed and swelled by gradually pouring 500 mL of 1 mM HCl over the resin, and further washed with 100 mL of water and then with 100 mL of 10 mM potassium phosphate, pH 8.0. The resin was immediately transferred to a 15-mL polypropylene tube and 10 mM potassium phosphate (pH 8.0) was added (*1 mL) until the resin was a thick slurry. The purified 144 bp oriA PCR product was immediately added to the resin and incubated on a rotating wheel, end-overend, at 25°C overnight ([8 h), transfered to a ceramic funnel with a glass fiber filter, and washed with two 100 mL washes of water and one 100 mL wash of 1 M ethanolamine hydrochloride, pH 8.0. After transferring to a 15-mL polypropylene tube, 1 M ethanolamine hydrochloride (pH 8.0), was added (*1 mL) until the resin was a smooth slurry. The resin was incubated on a rotating wheel, end-over-end, for 2–4 h at room temperature, transfered to a ceramic funnel with a glass fiber filter, and washed with 100 mL of 10 mM potassium phosphate, pH 8.0, 100 mL of 1 M potassium phosphate, pH 8.0, 100 mL of 1 M KCl, 100 mL of water, and 100 mL of column storage buffer (10 mM Tris–Cl, pH 7.8, 1 mM EDTA, pH 8.0, 0.3 M NaCl, and 0.04% (w/v) sodium azide). The resin was transfered to a 15-mL polypropylene tube and stored in *5 mL column storage buffer at 4°C. Isolation of origin binding protein activity
Fig. 1 Detection of ori-binding proteins from total chloroplast extracts via southwestern blot. Lane 1 was probed with pUC19 DNA; lane 2 was probed with a 168 bp fragment of oriA; lane 3 was probed with a 1.3 kb fragment containing oriA; lane 4 was probed with a 248 bp fragment or oriB; lane 5 was probed with a 507 bp fragment containing oriB (See Lugo et al. 2004 for details of ori fragments)
Total chloroplast proteins were loaded onto an equilibrated DEAE cellulose column (*70 cm3 column volume) at 0.25 mL/min using a BioLogic LP chromatography system (Bio-Rad). The column was washed with 500 mL buffer A (50 mM Tris–Cl, pH 8.0, 10 mM b-mercaptoethanol, 0.2 mM phenylmethyl sulfonyl fluoride, 20% glycerol, 10 mM sodium metabisulfite, 10 mM benzamidine, 50 mM KCl) at 0.50 mL/min and then eluted in 4 mL fractions with 130 mL buffer A/0.6 M KCl at 0.33 mL/min. OriA binding activity was detected via electrophoretic mobility shift assay. Active fractions were pooled and dialyzed in buffer Z (50 mM Tris–Cl, pH 7.8, 12.5 mM MgCl2, 1.0 mM dithiothreitol, 20% glycerol, 0.1% Nonidet P-40, 100 mM KCl) overnight with 2 buffer changes. Poly(dI/dC) (10 lg/mL) was added to the dialysate to bind non-specific proteins, and the mixture was loaded onto an equilibrated oriA-coupled Sepharose column from above (*5.5 cm3
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column volume, coupled with 144 bp oriA fragment) at 0.25 mL/min. The column was washed with 10 mL of buffer Z at 0.33 mL/min or with four 2 mL aliquots of buffer Z at gravity flow, carefully washing the sides of the column. The bound proteins were eluted with 10 mL of a linear gradient of buffer Z from 0.1 M KCl to 0.9 M KCl at 0.25 mL/min, followed by 5 mL of buffer Z/1.0 M KCl at 0.25 mL/min, and then with 5 mL of buffer Z/2.0 M KCl at 0.25 mL/min. 1 mL fractions were collected and oriA binding activity was detected via electrophoretic mobility shift assays. Electrophoretic mobility shift assays Electrophoretic mobility shift assays were performed using the LightShift Chemiluminescent EMSA Kit (Pierce) according to the manufacturer’s instructions with slight modifications. Each 20 ll reaction contained 2 lg poly (dIdC), 1X LightShift binding buffer, 10 mM MgCl2, 5–10 ll protein fraction, and 1 ll biotinylated oriA fragment. Reactions were incubated at room temperature for 30 min. All samples were loaded onto a 5% TBE gel and run in 0.5X TBE at 75 V until the tracking dye traveled * 3/4 of the way through the gel. DNA was transferred to a nylon membrane in a tank transfer apparatus (Bio-Rad) at 380 mA for 45 min in cold 0.5X TBE. The nylon membrane was cross-linked at 150 m Joules/cm2 in a GS Gene Linker (Bio-Rad). Biotinylated oriA was detected using the LightShift Chemiluminescent EMSA kit according to the manufacturer’s instructions. SDS–PAGE analysis OriA affinity column fractions were concentrated by acetone precipitation and analyzed by SDS–PAGE. Pellets were resuspended in 20 ll of 2X SDS-sample loading buffer and heated at 95°C for 5 min. Samples were then loaded onto a 8–16% Tris–glycine gel (Invitrogen) and run at 30 mA in 1X SDS-running buffer until the tracking dye reached the bottom of the gel. The gel was washed 3 times in dH2O for 5 min, stained with Bio-Safe Coomassie (BioRad) for 1 h, and destained overnight in dH2O. MALDI-ToF mass spectrometry Protein bands for mass spectrometry analysis were excised from 8–16% Tris–glycine gels (Invitrogen) under sterile conditions and transferred to clean 1.5 mL microcentrifuge tubes. Excised gel bands were stored at –20°C. In-gel tryptic digests of protein bands were performed according to Shevchenko et al. (1996) with slight modifications. 1.5 mL of 50% acetonitrile/50 mM ammonium bicarbonate was added to each tube containing a gel band. Samples
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were placed into a micro mixer and rotated for 30 min at room temperature. The liquid was then aspirated off, and 140 lL of 100 mM ammonium bicarbonate was added to the samples, followed by 10 lL of reduction buffer (75 mM dithiothreitol, 100 mM ammonium bicarbonate). Samples were incubated with no motion at 60°C for 45 min. Samples were then removed from the heat block, cooled to room temperature, and solutions were aspirated off. 140 lL of 100 mM ammonium bicarbonate was added to the samples, followed by 10 lL of alkylation buffer (0.3 M iodoacetamide, 100 mM ammonium bicarbonate). Samples were incubated in the dark at room temperature for 30 min, and the liquid was aspirated off. 1 mL of 100 mM ammonium bicarbonate was added to each gel spot and tubes were rotated at room temperature for 30 min. Solutions were then aspirated off. Under sterile conditions, gel bands were cut 1–2 times (depending on size of gel spot) with a sterile razor blade and transferred to a clean 1.5 mL microcentrifuge tube. 500 lL of 50% acetonitrile/50 mM ammonium bicarbonate was added to each sample tube and rotated at room temperature for 30 min, followed by removal of the solution. Gel pieces were dried to completeness in a speed vac. 10 lL of trypsin digestion solution (0.02 mg/mL trypsin in 25 mM ammonium bicarbonate) was added directly to each gel sample and incubated for 10 min at room temperature. This step was repeated until the gel pieces were completely hydrated. 25 mM ammonium bicarbonate (usually 10–20 lL) was added to the samples to have a 0.5 mm excess of solution above the gel pieces. Samples were incubated at 37°C with rocking for 16–24 h. Following incubation, 1 lL of 88% formic acid was added to the samples to stop the trypsin reaction. Samples were sonicated for 20 min in a water bath and then centrifuged briefly. Samples were concentrated and desalted using ZipTips (Millipore). Mass spectrometry analysis was performed on an API QSTAR Pulsar I (Applied Biosystems). Protein identifications were made using the nonrestricted NCBI protein database. Western blot analysis Chloroplasts and mitochondria were purified as described (Khazi et al. 2003) from young soybean seedlings. The purified organelles were lysed in 1X SDS–PAGE loading buffer and the proteins were electrophoresed in a 4–20% polyacrylamide gel (Sambrook and Russell 2001), followed by transfer of the proteins to PVDF membrane. An antibody against the Arabidopsis SSB2 protein (Edmondson et al. 2005) was incubated with the membrane followed by washing and incubation with second antibody conjugated with horseradish peroxidase (HRP) as described (Khazi et al. 2003). Control antibodies for chloroplast protein (large subunit of ribulose carboxylase) and mitochondria
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(glycine decarboxylase) were used in separate identical blots for identification of organelle localization. After the final washes, membranes were incubated in Pierce SuperSignal West Pico Substrate for 5 min, wrapped in plastic wrap and exposed to film.
Results Southwestern blot analysis Total chloroplast proteins were separated by SDS–PAGE, transferred to a membrane, and incubated with radioactively labeled DNA fragments specific for the oriA and oriB regions of ctDNA. For all four ori-specific fragments two protein bands of approx. 28 and 20 kDa showed interaction with the probes (Fig. 1 lanes 2–5). For the control lane incubated with pUC19 vector DNA, a single protein band was observed of about 42 kDa (Fig. 1 lane 1), which was also visualized in each of the other lanes, suggesting non-specific DNA binding for this protein. The ability to identify a sequence-specific binding protein(s) led us to proceed with isolation of the origin-binding protein(s).
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OriA-affinity chromatography Active fractions from the DEAE-cellulose column were pooled and subjected to further purification through an orispecific affinity column. Fractions eluted from the affinity column were separated by SDS–PAGE (Fig. 3a). One distinct protein band at *28 kDa was eluted at high salt. It is notable that the molecular weight is close to the 28 kDa chloroplast protein that was identified by southwestern blot analysis in Fig. 1. Importantly, this protein band was not present in a control column (no oriA coupled to the resin) run under the same conditions (Fig. 3b). The 28 kDa band was excised from the gel for MALDI-ToF mass spectrometry analysis.
Detection of OriA-binding proteins in cellulose column fractions Electrophoretic mobility shift assays were performed to identify chloroplast fractions containing oriA-binding activity. A shift of the 144 bp oriA fragment was observed with chloroplast protein fractions eluted from DEAE cellulose (Fig. 2). Two major shifts appear in the fractions loaded in lanes 3–6. The observation of a double shift could be due to multiple binding sites in the fragment or to the presence of multiple proteins that bind the fragment.
Fig. 2 Electrophoretic mobility shift assay of DEAE cellulose column fractions. Lane 1 is a kit positive control. Lane 2 contains no protein. Lanes 3–12 contain column fractions. Arrow pointing to the right indicates a shift in the kit control. Arrows pointing to the left indicate shifts of oriA. Free oriA is marked as probe
Fig. 3 OriA affinity column fractions. The lane on the far left of each gel is the marker; the second lane from the far left of the gel on the left is the total dialyzed protein loaded onto the column. a lanes 1–7, 9, 11, 13, 15, 17, and 19 refer to the affinity column fraction loaded. Arrow indicates the 28 kDa protein band. b CNBR-activated Sepharose resin was prepared identically as the oriA-coupled affinity resin, except that no oriA was added to the resin. Lane 1 is a marker. Lane 2 is the last wash fraction. Lanes 3–11 are even fractions 4–20. The arrow indicates approximately where 28 kDa is on the gel
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Sequence analysis of the 28 kDa soybean protein The 28 kDa protein band was digested with trypsin for analysis by MALDI-ToF mass spectrometry. Seven strong peaks were chosen for MS/MS analysis, and all seven produced comparable MS/MS spectra. The resulting data were subjected to BLAST searches of the nonredundant NCBI protein database. BLAST results from three peaks identified a 25,897 Da soybean protein, encoded by the C6SY33 gene (accession number ACU14156; hereafter to be referred to as the 26 kDa protein). Similarity of a large portion of this protein to an Arabidopsis thaliana singlestranded binding protein targeted to mitochondria (mtSSB) was also identified. BLAST searches of the Arabidopsis and soybean genomes produced no additional homologues within these two species. Further BLAST analysis identified two rice homologues (accession numbers NP_001056004 and EEC79506) and homologues from Ricinus communis (XP_002529271), Populus trichocarpa (XP_002319495) and Vitis vinifera (XP_002278555) of the 26 kDa protein. Each of these proteins are annotated as either unknown function proteins or proteins with putative single-stranded DNA binding activity. The ACU14156 soybean protein sequence was aligned with the Arabidopsis mtSSB protein, showing limited alignment outside the DNA binding domain of the protein, with additional sequences at both the N-terminal and C-terminal regions, creating a longer protein than the 21 kDa Arabidopsis protein (Fig. 4A). The soybean protein has limited homology with the E. coli SSB protein (Fig. 4B), suggesting it may have a different or additional function. While the soybean protein contains an OBF motif found in all SSB proteins, this motif is not limited to SSB proteins and is found in other DNA binding proteins as well, as found by a conserved domain search on the NCBI website (www.ncbi.nlm.nih.gov/structure/cdd). The exact role of the mtSSB protein in mitochondria is not known at this time, and it may play a regulatory role in addition to its function as an SSB protein, which is the only activity the Arabidopsis protein has been shown to have as yet (Edmondson et al. 2005).
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Silva-Filho 2003; Mackenzie 2005). It is possible that the soybean protein is a homologue to the Arabidopsis mtSSB and that the protein localizes to both mitochondria and chloroplasts. However, the Arabidopsis mtSSB protein was shown to localize exclusively to mitochondria by analysis of N-terminal sequence-GFP fusions in transformed plants (Edmondson et al. 2005), and is considerably smaller than the soybean protein. A second SSB homologue was identified in the Arabidopsis genome that was predicted to target mitochondria (Edmondson et al. 2005), but this protein has not been previously characterized. These Arabidopsis SSB proteins share high homology with each other and with the soybean 28 kDa protein. Antibodies against the second SSB homologue (termed SSB2 in our lab) have been shown in western blots to cross-react with a soybean chloroplast protein of the predicted size of 28 kDa (Fig. 5 lane 2). This antibody does not appear to cross-react with any protein in a purified mitochondrial fraction (Fig. 5 lane 3). Purity ([98%) of the organelle fractions was confirmed using control antibodies against a chloroplast protein (RbcL) and glycine decarboxylase (data not shown). DNA binding activity of the 28 kDa protein Electrophoretic mobility shift assays were performed with fractions containing the 28 kDa protein using the 144 bp oriA fragment as probe (Fig. 6). Fraction 4 displays no shift (lane 2), while strong shifts are observed in both fractions 14 and 15 (lanes 4 and 5), which contain the purified and concentrated 28 kDa protein. Importantly, mobility shift assays with purified Arabidopsis mtSSB (overexpressed in the mature form in E. coli and purified on a nickel-chelate column; Edmondson et al. 2005) performed under the same conditions do not result in a shift of oriA (lane 6), but do result in a shift of single-stranded DNA (ssDNA) (Figure 7). This indicates that the 28 kDa soybean protein and Arabidopsis mtSSB differ in their activity, as only the soybean protein causes a shift of double-stranded oriA, and it does not bind nonspecific ssDNA (Fig. 7).
Discussion Organellar localization of the soybean origin-binding protein Analysis of the amino acid sequence of the 28 kDa protein by some targeting prediction programs suggest this protein is localized to chloroplasts (WoLFPSORT and ChloroP) while other programs suggest mitochondrial localization (TargetP, MitoProt II and Predotar). Although many organelle-targeted proteins localize to a single target, dualtargeted proteins have been identified that localize to both mitochondria and chloroplasts (Peeters and Small 2001;
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A*28 kDa soybean protein was isolated by ctDNA replication origin sequence specific affinity chromatography from total soybean chloroplast proteins. Mass spectrometry analysis identified the gene as C6SY33 in the soybean genome, which is annotated as a putative uncharacterized gene with a molecular weight of 25,897 Da. This soybean protein shares some homology within an SSB domain of an Arabidopsis mitochondrial-targeted SSB (mtSSB) of 21 kDa, but is considerably longer with the extra sequence at both the N-terminal and C-terminal ends. Electrophoretic
Plant Mol Biol (2011) 76:463–471 Fig. 4 Sequence alignments of the soybean origin-binding protein. A, alignment of the soybean protein with the Arabidopsis mtSSB protein (Edmondson et al. 2005). B, alignment of the soybean protein with E. coli SSB. Identical amino acids between the two proteins are indicated by a vertical line; very similar amino acids by a colon (:), and similar amino acids by a period (.)
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(A) Soybean OBP mtSSB Soybean OBP mtSSB Soybean OBP mtSSB Soybean OBP mtSSB Soybean OBP mtSSB
1 MNSMAVRLVKHLQLS--APTSSSFGGVPRSGASWYSTSLSGGEYQPSQDN |||:|:|:.|.|:.| :|.:.| ..|...||:|| 1 MNSLAIRVSKVLRSSSISPLAIS---AERGSKSWFST-------------
48
49 VAPKNEALDGELDDFLGEKAELQLQGVDPKKGWGFRGVHKAIICGKVGQA .|.:|.::.:.::.:.|:.|||..||||:|||||||||:|||||||||| 35 -GPIDEGVEEDFEENVTERPELQPHGVDPRKGWGFRGVHRAIICGKVGQA
98
99 PVQKILRNGRNLTIFTVGTGGMFDQRIQGPKDLPKPAQWHRIAVHNDILG |:||||||||.:||||||||||||||:.|..:.|||||||||||||::|| 84 PLQKILRNGRTVTIFTVGTGGMFDQRLVGATNQPKPAQWHRIAVHNEVLG
148
149 AYAVQKLFKNSSVYVEGDIEIRVYNDSINGEVKSIPEICVRRDGKICLIK :||||||.||||||||||||.|||||||:.||||||||||||||||.:|| 134 SYAVQKLAKNSSVYVEGDIETRVYNDSISSEVKSIPEICVRRDGKIRMIK
198
199 SGESIDKTSLDELRERVVLGKSDHLTEAYTSVRNFTLL .||||.|.|.|||:|.:: 184 YGESISKISFDELKEGLI--------------------
34
83
133
183
236 201
(B) Soybean OBP
1 MNSMAVRLVKHLQLSAPTSSSFGGVPRSGASWYSTSLSGGEYQPSQDNVA
50
E. coli SSB
0 --------------------------------------------------
0
Soybean OBP
51 PKNEALDGELDDFLGEKAELQLQGVDPKKGWGFRGVHKAIICGKVGQAPV ...|||:|.|:.|.:||.|. 1 ------------------------------MASRGVNKVILVGNLGQDPE
100
101 QKILRNGRNLTIFTVGTGGMFDQRIQGPKDLPKPAQWHRIAVHNDILGAY .:.:.||..:...|:.|...:..:..| ::.:..:|||:.:... |... 21 VRYMPNGGAVANITLATSESWRDKATG--EMKEQTEWHRVVLFGK-LAEV
150
151 AVQKLFKNSSVYVEGDIEIRVYNDSINGEVKSIPEICVRRDGKICLIKSG |.:.|.|.|.||:||.:..|.:.|. 68 ASEYLRKGSQVYIEGQLRTRKWTDQ-------------------------
200
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E. coli SSB
201 ESIDKTSLDELRERVVLGKSDHLTEAYTSVRNFTLL-------------.|:..:.||...:|.....: 93 ----------------SGQDRYTTEVVVNVGGTMQMLGGRQGGGAPAGGN
Soybean OBP
236 --------------------------------------------------
236
E. coli SSB
127 IGGGQPQGGWGQPQQPQGGNQFSGGAQSRPQQSAPAAPSNEPPMDFDDDI
176
Soybean OBP
236 --
236
E. coli SSB
177 PF
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E. coli SSB Soybean OBP E. coli SSB Soybean OBP E. coli SSB Soybean OBP
Fig. 5 Western blot to show localization of the soybean 28 kDa protein to chloroplasts. Purified soybean chloroplasts and mitochondria were lysed and proteins were separated by SDS–PAGE as described in Materials and Methods. An antibody against the Arabidopsis SSB2 protein was used. Lane 1, molecular weight markers (Pierce magic markers, sizes are indicated in the left margin); lane 2, soybean chloroplast protein fraction; lane 3, soybean mitochondrial protein fraction; lane 4, total soybean proteins
mobility shift assay data indicate that there are distinct differences between these two proteins. The Arabidopsis mtSSB appears to be targeted to mitochondria exclusively
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Fig. 6 OriA electrophoretic mobility shift assays with oriA affinity column fractions. Lane 1 contains no protein; lane 2 contains oriA affinity fraction 4; lane 3 contains oriA affinity fraction 5; lane 4 contains oriA affinity fraction 14; lane 5 contains oriA affinity fraction 15; lane 6 contains Arabidopsis mtSSB
(Edmondson et al. 2005). The soybean ctDNA ori-binding protein reported here is predicted to target chloroplasts by some programs and to target mitochondria by others, and it is possible that it may be dual-targeted. The only close sequence similarity found when performing a Blast search with the soybean sequence against
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Fig. 7 Single-stranded DNA electrophoretic mobility shift assay with the 28 kDa soy protein and Arabidopsis mtSSB. Lane 1 contains no protein; lanes 2–5 contain the 28 kDa protein (oriA affinity fraction 15) with decreasing amounts of ssDNA; lane 6 contains Arabidopsis mtSSB
the plant sequence database is with two Arabidopsis mtSSB homologues, and with some putative proteins of unknown function in a few other species. However, mobility shift assays performed with the 28 kDa soybean protein and Arabidopsis mtSSB (the SSB reported in Edmondson et al. (2005); we have designated this as mtSSB1) show differences in their binding abilities. Western blot analysis using antibody against the Arabidopsis SSB2 homologue indicates that the 28 kDa protein is highly enriched in concentrated soybean chloroplast fractions, while the mtSSB1 homologue was previously shown by GFP fusion protein localization to target mitochondria exclusively (Edmondson et al. 2005). These results suggest that the two Arabidopsis SSB homologues are localized to separate organelles. We have not been able to identify two separate homologues in the soybean genome. It is thus unclear at present whether the soybean 28 kDa protein is localized only to chloroplasts or may also be localized to mitochondria, although this was not detected in our western blots. The 28 kDa protein shifts a double-stranded oriA fragment while Arabidopsis mtSSB fails to shift oriA (Fig. 6). Arabidopsis mtSSB in contrast causes a strong shift of ssDNA but cannot shift the ori fragment (Fig. 7). The sequence homology identified with the Arabidopsis mtSSB (Fig. 4A) is restricted to a portion of a conserved SSB domain. The soybean protein also has limited homology with the E. coli DnaA protein, the initiator protein for
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bacterial chromosomal replication. It is possible that the SSB domain homologous to both the 28 kDa soybean protein and the Arabidopsis mtSSB is common among a variety of plant DNA binding proteins. Further work is needed to determine the extent of sequence-specific and random DNA binding proteins in other plant species. Two-dimensional gel electrophoresis of the 28 kDa protein suggests the possibility that this protein may undergo post-translational modification such as phosphorylation (data not shown), which is known to modify the activity of some regulatory proteins. If the 28 kDa protein is involved in ctDNA replication initiation, posttranslational modifications may be a way of regulating or modifying its activity. The isoelectric point (pI) of the 28 kDa protein is between 5.5 and 6.0 (data not shown). DNA binding proteins are typically more basic in their charge. However, E. coli SSB has a pI of 6.0, and Synechococcus SSB has a predicted pI of 5.0 (Meyer and Laine 1990; Synechococcus SSB pI predicted by EMBL WWW Gateway to Isoelectric Point Service, http://www.embl-heidel). The C-terminus of E. coli SSB, which does not appear to play a direct role in DNA binding, contains several negatively charged amino acids that when cleaved leaves the SSB with a pI of 8.9 (Kinebuchi et al. 1997; Meyer and Laine 1990). Thus, while the 28 kDa protein has an overall pI of between 5.5 and 6.0, it may contain DNA-binding domains with a much more basic pI. Also, it is worth noting that the predicted pI of the mature Arabidopsis mtSSB is 8.2, which is significantly different than that of the 28 kDa protein (mtSSB pI predicted by EMBL WWW Gateway to Isoelectric Point Service, http://www.embl-heidel). This protein may play a specific role in ctDNA D-loop replication through initial binding of the ori regions and facilitating the assembly of the replication apparatus similar to the role of the DnaA protein in E. coli. However, other proteins may be involved in the initiation of replication from other replication origins in the chloroplast genome, including those for rolling circle replication and recombination-mediated replication. Recently, a chloroplast-localized RecA protein has been shown to play a significant role in ctDNA maintenance, as a mutation in this gene leads to a reduction in ctDNA copy number, reduced chloroplast function and to plant distress (Rowan et al. 2010). In summary, these results suggest that the soybean C6SY33 (accession number ACU14156) protein interacts specifically with the chloroplast oriA sequence and therefore may play a role in ctDNA replication initiation. While this protein shows some homology to Arabidopsis mtSSB, there are some critical differences in the predicted localization and activity of these proteins in vitro, suggesting that they have different functions in vivo. Further work is needed to determine whether this protein may possibly be
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dual targeted and play a role in both ctDNA and mtDNA replication. The interactions of this protein with other chloroplast proteins will be helpful in determining the role that this protein may play in ctDNA replication. Acknowledgments We thank Dr. Craig Thulin and Katie Southwick for assistance with the mass spectrometry and 2-D gel analysis, and Luis Alvarez, Johnathon Overson, Colton Kempton and Cindee Perry for assistance with some of the experiments. This work was supported by grants from the USDA and from the BYU Mentoring Environments Grant program.
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