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Journal of Bacteriology, September 2006, p. 6153-6167, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00045-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Methanosarcina acetivorans Flap Endonuclease 1 Activity Is Inhibited by a Cognate Single-Stranded-DNA-Binding Protein{dagger}

Yuyen Lin,1 Claudia E. Guzman,1 Mary C. McKinney,2 Satish K. Nair,3 Taekjip Ha,2,4,5 and Isaac K. O. Cann1,4,6*

Department of Animal Sciences,1 Department of Physics,2 Department of Biochemistry,3 Institute for Genomic Biology,4 Howard Hughes Medical Institute,5 Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 618016

Received 11 January 2006/ Accepted 11 June 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms, Methanosarcina acetivorans harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5' region cleaved during Okazaki fragment processing. We report here that M. acetivorans RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by M. acetivorans flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Single-stranded-DNA-binding proteins (SSB) in the bacterial lineage and replication protein A (RPA) in the archaeal/eukaryotic sister lineages are proteins required for diverse DNA transactions, including replication, repair, and recombination (16, 26). Central to the structure of SSB and RPA proteins is the so-called oligonucleotide/oligosaccharide-binding (OB) fold, a small motif usually ranging from 70 to 150 amino acids (18). Structurally, OB folds consist of a pair of three antiparallel ß-sheets, although in many cases, the first ß-sheet is shared by the two groups (18). Between the third and fourth strands is usually an {alpha}-helix. The large variation in length of the OB folds is primarily due to the length of the variable loops found in their conserved secondary structural elements (24) (Fig. 1A).


Figure 1
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  		FIG. 1. Oligonucleotide/oligoscaccharide binding folds in MacRPA1. (A) Structure-based model of an archaeal OB fold domain such as observed in replication protein A from M. acetivorans. The common fold consists of a five-stranded ß-sheet that twists to form a closed barrel and is capped by a short {alpha}-helix. In this diagram, the ß-sheets are orange and the single helix is blue, and both termini are labeled. This figure was generated using the Ribbons software (4). (B) A schematic representation of MacRPA1 wild type and its truncated forms MacRPA1{Delta}C1, MacRPA1{Delta}C2, MacRPA1{Delta}C3, MacRPA1{Delta}C1{Delta}N1, MacRPA1{Delta}N1, MacRPA1{Delta}N2, and MacRPA1{Delta}N3 showing their respective OB folds (the shaded boxes with letters A, B, C, and D). The motifs are not drawn to scale. The arrows above and below each box show the forward and reverse primers used to amplify the DNA fragments coding for each MacRPA1 derivative.

 
The bacterial SSB, in general, is composed of a single OB fold, although in solution the protein oligomerizes into a homotetramer (16). Recently, a new form of bacterial SSB that harbors two OB folds in a single polypeptide was isolated from Deinococcus radiodurans. This protein forms a homodimer to mimic the homotetrameric bacterial SSB (2). The eukaryotic RPA, on the other hand, is composed of three different proteins (RPA70, RPA32, and RPA14) that form a heterotrimeric complex (26). Eukaryotic RPA interacts with polymerase {alpha}-primase complex and therefore participates in initiation of DNA replication and also synthesis of each Okazaki fragment (1, 8, 17, 19).

Most bacteria harbor the Escherichia coli SSB type, whereas most eukaryotes have the heterotrimeric type of RPA. In contrast, members of the archaea harbor different forms of single-stranded DNA (ssDNA) binding proteins that are more similar to their eukaryotic counterpart at the amino acid sequence level (9, 11-14). The archaeal RPA proteins are either single polypeptides (9, 11, 12, 25) or heterocomplexes of more than one protein (5, 14).

In the mesophilic archaeon Methanosarcina acetivorans we demonstrated the presence of three functional RPA proteins (22), which are also conserved in its relatives Methanosarcina mazei and Methanosarcina barkeri. Two of the RPA proteins were of similar architecture, in that both proteins (M. acetivorans RPA2 [MacRPA2] and MacRPA3) have two OB folds within the N-terminal half and a zinc finger domain in the C-terminal half (22). The MacRPA1 protein, in contrast, is a large polypeptide composed of four OB folds, and unlike previously described archaeal RPA proteins of similar architecture, it lacks a zinc finger motif (22). Thus, the RPA proteins from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus are composed of four and five OB folds, respectively, in the same polypeptide, but in addition they also contain putative zinc finger motifs at the C terminus (11, 12).

Due to the conservation of the three MacRPA proteins in the Methanosarcinales, we were interested in investigating, through in vitro analysis, whether the individual proteins have unique properties that may lead to differences in effects on processes that require ssDNA-binding proteins. Archaea and eukaryotes are known to harbor related DNA replication proteins (3), and of particular interest to us was lagging-strand DNA synthesis, since it was recently shown that in eukaryotes RPA regulates the size of the flap structure cleaved from the 5' region during Okazaki fragment maturation (1). Thus, we compared the effects of the three different RPA homologs from M. acetivorans on the processing of a flap substrate by M. acetivorans flap endonuclease I (MacFEN1). Interestingly, only MacRPA1 strongly inhibited the activity of MacFEN1, and to gain insights into the contribution of the different regions of MacRPA1 to its inhibitory activity, N-terminal and C-terminal truncated derivatives of the protein were created and analyzed for their capacity to bind to ssDNA and also to inhibit the endonuclease activity of MacFEN1.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cloning of genes. The cloning of the genes encoding MacRPA1, MacRPA2, and MacRPA3 was described in our previous report (22). MacRPA1 is made up of four OB folds, and to investigate the contribution of the individual OB folds to ssDNA-binding and the functional property under study, we designed primers for a sequential deletion of individual OB folds from the N terminus and also from the C terminus. All PCR primers used in this report are presented in Table 1. The forward primer, MacRPA1F, was combined with the reverse primers {Delta}C1-R, {Delta}C2-R, and {Delta}C3-R to amplify M. acetivorans rpa1 derivatives encoding products with one, two, and three OB folds deleted, respectively, from the C terminus. The plasmid constructs for expression of the three C-terminally truncated MacRPA1 derivatives were designated pET28/rpa1{Delta}c1, pET28/rpa1{Delta}c2, and pET28/rpa1{Delta}c3, and the corresponding products are referred to as MacRPA1{Delta}C1, MacRPA1{Delta}C2, and MacRPA1{Delta}C3, respectively (Fig. 1B). A forward primer, {Delta}N1-F, and the reverse primer {Delta}C1-R were combined to create a MacRPA1 derivative with the first (Fig. 1B, fold A) and the last (Fig. 1B, fold D) OB folds deleted, thus leaving a protein comprising the two middle OB folds. The plasmid construct was designated pET28/rpa1{Delta}c1{Delta}n1, and its product is referred to as MacRPA1{Delta}C1{Delta}N1. Next, three N-terminally truncated derivatives of MacRPA1 were made by combining the reverse primer, MacRPA1R, with the forward primers {Delta}N1-F, {Delta}N2-F, and {Delta}N3-F. The three plasmid constructs were designated pET28/rpa1{Delta}n1, pET28/rpa1{Delta}n2, and pET28/rpa1{Delta}n3, and their products are designated MacRPA1{Delta}N1, MacRPA1{Delta}N2, and MacRPA1{Delta}N3, respectively. The DNA template for the PCR amplification was a pET28 plasmid construct containing the gene for MacRPA1 wild type (22). We also cloned the gene for a putative Flap endonuclease 1 (MacFEN1; GenBank accession number AAM07354) from M. acetivorans. The gene was amplified by using the two oligonucleotides MacFen1F and MacFen1R as the forward and reverse primers, respectively, and M. acetivorans genomic DNA was used as the template. The PCR product was inserted into pET28a plasmid, and the construct was designated pET28/fen1. The product from this construct will be referred to as MacFEN1. Each forward primer incorporated an NdeI site to facilitate fusion of the amplified gene to a six-histidine (His6) encoding sequence in the vector used for gene expression (pET28a; Novagen). Thus, to create the expression vectors, each PCR product was initially cloned into a TA cloning vector (pGEMT vector; Promega) and then digested with the appropriate restriction enzymes, followed by insertion into a modified pET28a plasmid digested with NdeI and XhoI. The modification of the pET28a plasmid was the replacement of the kanamycin resistance gene with that for ampicillin resistance. Each insert in the plasmid constructs was sequenced (W. M. Keck Center for Functional and Comparative Genomics, University of Illinois at Urbana-Champaign) to ensure the integrity of the DNA sequence.


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  		TABLE 1. Oligonucleotides used in this studya

 
Production of recombinant proteins. Recombinant MacRPA1 wild type, truncated MacRPA1 proteins, and MacFEN1 were expressed in E. coli BL21-CodonPlus(DE3) RIL cells (Stratagene). An E. coli cell line for each individual protein was made by transforming the respective expression vector into E. coli cells by the heat shock method. The product of each transformation was spread on an LB plate supplemented with ampicillin (100 µg/ml) and chloramphenicol (50 µg/ml). The plates were then incubated at 37°C overnight. A single colony from each cell line was inoculated into LB broth supplemented with the two antibiotics, and the cells were cultured at 37°C until the optical density at 600 nm reached 0.3. Gene expression was then induced by adding isopropyl-ß-D-thiogalactopyranoside to the culture at a final concentration of 0.1 mM. After a further 12 h of culturing at 16°C, the cells were harvested by centrifugation. The cell pellets were suspended in a lysis buffer (50 mM sodium phosphate, pH 7.0, 300 mM NaCl), and cell contents were released by lysis with a French pressure cell (American Instruments Co.). After the cell debris was removed by centrifugation (10,000 x g for 20 min at 4°C), the supernatant was applied to a lysis buffer-equilibrated metal affinity resin (TALON cobalt affinity resin; Clontech). The protein-bound resin was washed with 10 column volumes of lysis buffer, and the protein that bound to the resin was eluted with an elution buffer (lysis buffer containing 150 mM imidazole). For further purification of MacRPA1 and its truncated derivatives, the fractions were pooled and dialyzed against a buffer containing 50 mM sodium phosphate, pH 7.0, and 150 mM NaCl. The dialysate was applied to a gel filtration column (Superdex 200HR 10/30 column; Amersham Biosciences), and the chromatography was performed with the same buffer at a flow rate of 0.5 ml/min at 4°C. The highly purified proteins were dialyzed against a buffer containing 50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 10% glycerol, and 0.5 mM dithiothreitol (DTT) for storage. In the case of MacFEN1, after elution from the affinity resin, the protein was dialyzed against a buffer composed of 50 mM Tris-HCl, pH 8.0, and 100 mM NaCl. The dialysates were loaded onto an anion exchange column (HiTrap Q-XL; Amersham Biosciences) preequilibrated in the same buffer and fitted to a high-pressure liquid chromatography column (AKTA Explorer 10; Amersham Biosciences). The column was washed with 10 column volumes of the equilibration buffer, and then the bound proteins were eluted with a buffer containing 50 mM Tris-HCl, pH 8.0, and 1 M NaCl. The eluted proteins were dialyzed against the storage buffer described above for the RPA proteins.

Subunit organization. The subunit organizations of the truncated MacRPA1 proteins were estimated by size exclusion chromatography. Each truncated protein (100 µg) in 100 µl of a buffer containing 50 mM sodium phosphate, pH 7.0, and 150 mM NaCl was applied to a gel filtration column (Superdex 200HR 10/30; Amersham Biosciences) equilibrated in the same buffer. The chromatography was developed with the same buffer at a flow rate of 0.5 ml/min at 4°C, and the absorbance at 280 nm was monitored. Fractions (volume, 500 µl) were collected, and aliquots were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE). To calibrate the column, a set of protein standards [ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and RNase A (13.7 kDa) (Amersham Biosciences)] was analyzed under the same conditions, and the results were used in generating a standard curve for estimation of the relative molecular mass of each truncated derivative of MacRPA1.

Endonuclease assay. The annealing reactions to create the flap substrates were performed as follows: 20 pmol of 32P-end-labeled 34-mer oligonucleotide (oligonucleotide 1) was mixed with 20 pmol of an unlabeled 16-mer oligonucleotide (oligonucleotide 2) and 20 pmol of a 30-mer unlabeled oligonucleotide (oligonucleotide 3). Oligonucleotide 3 was fully complementary to oligonucleotide 2 and partially complementary to oligonucleotide 1 (Fig. 2A, i). The annealing reaction was carried out in 20 µl of a buffer containing 20 mM Tris-HCl, pH 8.0, and 100 mM NaCl. The three oligonucleotides were originally described by other investigators in a previous report (10). The mixture was heated at 95°C for 5 min and then slowly cooled to room temperature to allow annealing of the three oligonucleotides, which resulted in a 20-mer ssDNA region in the labeled oligonucleotide serving as a flap (Fig. 2A, i). The sequences of the oligonucleotides are shown in Table 1. To create a flap of shorter sequence, we mixed 20 pmol of a 32P-end-labeled 19-mer oligonucleotide (oligonucleotide 5), 20 pmol of an unlabeled 16-mer oligonucleotide (oligonucleotide 4), and 20 pmol of an unlabeled 30-mer oligonucleotide (oligonucleotide 6). The annealing reaction was the same as described above. The 16-mer oligonucleotide (oligonucleotide 4) was fully complementary to oligonucleotide 6, whereas the 19-mer oligonucleotide (oligonucleotide 5) was partially complementary to oligonucleotide 6, and therefore annealing resulted in a flap of five nucleotides (Fig. 2A, ii). The endonuclease assay was performed in a reaction buffer containing 20 mM Tris-HCl, pH 8.8, 15 mM MgCl2, 2 mM DTT, 0.05 µg/µl bovine serum albumin, and 1 pmol of the flap substrate. MacFEN1 was added at 25 pmol, and where the effects of the three RPA proteins (MacRPA1, MacRPA2, and MacRPA3) from M. acetivorans were tested, they were added in increasing amounts of 1.0, 2.5, 5.0, 7.5, and 10.0 pmol per reaction. In the case where truncated MacRPA1 derivatives were tested, the same amounts of protein were used. The final volume of each reaction mixture was 20 µl. The reaction mixtures were incubated at 37°C for 5 min, and to terminate the reactions, 4 µl of a stop solution (98% formamide, 1 mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue) was added to each mixture. The products were heated at 95°C for 5 min and resolved through electrophoresis on a 15% denaturing polyacrylamide gel containing 7 M urea. The running buffer was a 1x Tris-borate-EDTA buffer, and the products were visualized by autoradiography. The quantitation of products was carried out with a phosphorimager (BAS-1800II; Fuji).


Figure 2
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  		FIG. 2. (A) Schematic representation of the flap substrates used in this study. The 20-nucleotide-long flap substrate (i) and the 5-nucleotide-long flap substrate (ii) are represented. Oligo, oligonucleotide. (B) Recombinant His6-tagged wild-type proteins produced for this study were resolved on 12% sodium dodecyl sulfate-PAGE and stained with Coomassie brilliant blue. Lane 1, protein molecular mass marker (Fermentas); lane 2, MacFEN1; lane 3, MacRPA1; lane 4, MacRPA2; lane 5, MacRPA3. (C) The truncated derivatives of MacRPA1. Lane 1, protein molecular mass marker (Fermentas); lane 2, MacRPA1{Delta}C1; lane 3, MacRPA1{Delta}C2; lane 4, MacRPA1{Delta}C3; lane 5, MacRPA1{Delta}C1{Delta}N1; lane 6, MacRPA1{Delta}N1; lane 7, MacRPA1{Delta}N2; and lane 8, MacRPA1{Delta}N3.

 
EMSA. The capacities of MacRPA1 and its truncated derivatives to bind to ssDNA and double-stranded DNA (dsDNA) were analyzed by electrophoretic mobility shift assay EMSA. A total of 2 pmol of a 32P-end-labeled 42-mer oligonucleotide (Table 1, MacMC-R) was incubated with increasing amounts (1 to 5 pmol) of MacRPA1 or its truncated derivatives in 20 µl of binding buffer (20 mM Tris-HCl, pH 8.8, 15 mM MgCl2, 2 mM DTT, and 0.05 mg/ml bovine serum albumin) at 25°C for 30 min. To determine whether the MacRPA1 derivatives that bound to ssDNA can bind specifically to this substrate, their abilities to bind to ssDNA were challenged with 10x, 25x, and 50x cold ssDNA or cold dsDNA (Table 1, dsDNA). Two microliters of loading buffer (250 mM Tris-HCl, pH 7.5, 0.2% bromophenol, 40% glycerol) was added to the products of the reactions and resolved by electrophoresis on an 8% nondenaturing polyacrylamide gel in 1x Tris-borate-EDTA buffer. MacRPA1 wild type was also analyzed as a control. The results were visualized by autoradiography.

FPA. We used fluorescence polarization anisotropy (FPA), a technique that has been extensively applied in the detection of protein-DNA interactions, to estimate the dissociation constants (KD) of MacRPA1 and its truncated derivatives. FPA measures the rotational freedom of a fluorescent dye attached to a biomolecule by determining the intensity of polarized fluorescence as the polarized excitation is changed. The method has been described in detail in our previous reports (21, 22). Briefly, if the dye on the biomolecule rotates very quickly within its fluorescent lifetime, there will be little correlation between the excitation and the emission polarization. This will result in an anisotropy value close to zero. If the dye is rigidly attached, for example, in a situation where the biomolecule is bound by a protein, the emission polarization will reflect the excitation polarization, resulting in an anisotropy value close to the maximum of 0.4. A fast protein liquid chromatography-purified 18-base ssDNA tagged at the 5' end with fluorescein (FL-18; Operon Technologies) (Table 1) was used as the biomolecule. The ssDNA was in a reaction buffer composed of 20 mM Tris-HCl, pH 8.0, 15 mM MgCl2, and 2 mM DTT. Anisotropy was calculated according to the following equation: r = (IVV – GIVH)/(IVV + 2GIVH), where IVV and IVH are the vertically polarized emission intensities upon vertically polarized and horizontally polarized excitations, respectively. G is the correction factor for the equipment given as IHV/IHH, where IHV and IHH are the horizontally polarized emission intensities upon vertically polarized and horizontally polarized excitations, respectively. Thus, the anisotropy of the ssDNA alone will be low, and as any protein that binds to ssDNA is added, the value will increase and approach the maximum value (0.4). The FPA measurements were performed in a fluorometer at 23 ± 1°C (Cary Eclipse; Varian, Inc.); the reactions were excited at 490 nm, and emissions were measured at 518 nm. In order to estimate the dissociation constant, KD, and the Hill coefficients, n, each of the MacRPA1 derivatives was titrated in small increments until the anisotropy was saturated at a maximum value. The curves were then fitted with a Hill binding model using Origin 7 (OriginLab Corp.).

CD. The MacRPA1 wild type and its truncated derivatives were analyzed by circular dichroism (CD) spectroscopy to determine whether the deletions impacted the folding of the truncated proteins. After dialysis against a buffer composed of 50 mM Tris-HCl, pH 8.0, 75 mM NaCl, and 0.5 mM DTT, far-UV CD spectra of the recombinant proteins were determined. Each protein was at a concentration of 0.5 µg/µl, and the measurements were carried out at room temperature at a scanning rate of 50 nm/min from 200 to 260 nm by using a JASCO J-720 spectropolarimeter (Japan Spectroscopic Co.) and a cuvette (Starna) of path length 0.1 cm. Buffer runs were carried out to determine baseline readings, and all samples were baseline corrected before calculations. An average of triplicate scans for each sample was calculated in molar ellipticity and used to obtain the final data. The molar ellipticity ({theta}) was calculated based on the following equation: {theta} = ({theta}obs x 10–3 x MW)/(C x l x n x 10–2) degrees dmol–1 cm2, where {theta}obs is the observed ellipticity, MW is molecular weight, C is concentration (mg/ml), l is the path length of the cuvette in centimeters, and n refers to the number of residues (7).

DNA strand displacement by MacRPA proteins. Fluorescence resonance energy transfer (FRET) is a widely used "spectroscopic ruler" for measuring the distance between two fluorescent dyes attached to a biomolecule (22). Energy transfer efficiency can be defined as E = 1/[1 + (R/R0)6], where R is the distance between a donor and an acceptor fluorophore, and R0 is a characteristic constant depending on the particular dyes used (6). The DNA construct used in the unwinding experiments was a partial duplex DNA with a 20-base 3' tail and 18 bases of duplex DNA. The donor dye, Cy3, was internally labeled 9 bases into the short strand of DNA, and the acceptor dye, Cy5, was internally labeled 9 bases into the long strand of the DNA. Annealing of the two oligonucleotides brought the donor close to the acceptor dye, resulting in a high FRET signal. Thus, the FRET measured will only change if the duplex DNA is unwound at least 9 base pairs. By measuring the fluorescence intensity of both the donor and acceptor, one can approximate E by IA/(IA+ID), where IA is the acceptor emission intensity, and ID is the donor emission intensity, and we thereby infer the relative distance between the two dyes. Fluorescence measurements were performed in a Cary Eclipse (Varian Inc.) fluorometer at 23 (±1) °C with the same reaction buffer as that in the endonuclease experiments. Cy3 was excited at 515 nm, and fluorescence emission was collected at 565 nm (Cy3) and 665 nm (Cy5).

Amino acid sequence alignment. The amino acid sequence alignment was carried out with a multiple alignment program, ClustalW (http://www.ebi.ac.uk/clustalw/), and the shading was manually carried out. The amino acid sequences used for the alignment were retrieved from the protein database at the NCBI (http://www.ncbi.nlm.nih.gov/).

Protein concentration. The protein concentrations were determined by the Bradford method using a commercially available kit (Bio-Rad) with bovine serum albumin (New England Biolabs) as the standard.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of M. acetivorans RPA proteins on cleavage of flap DNA by MacFEN1. We reported earlier that each of the three different RPA proteins (MacRPA1, MacRPA2, and MacRPA3) in the archaeon M. acetivorans stimulated DNA synthesis by MacPolBI, a cognate DNA polymerase (22). The structural organization of MacRPA1 was different from that of MacRPA2 and MacRPA3, which shared a similar architecture of two OB folds and a zinc finger domain region. We reasoned that the different architectures of the RPA proteins may lead to differences in their roles in the cells of M. acetivorans. Aside from stabilizing ssDNA templates during DNA synthesis, ssDNA binding proteins participate in other DNA transactions, including Okazaki fragment processing (1). We therefore used in vitro analysis to determine the effects of the three RPA proteins on the endonuclease activity of a FEN1-like protein from M. acetivorans. The putative FEN1 shared 50% and 35% identities with Pyrococcus furiosus FEN1 and human FEN1, respectively, and we refer to it as MacFEN1. The gene for MacFEN1 was cloned and expressed, and the product was highly purified as shown in Fig. 2B, lane 2. In addition, the MacRPA proteins were produced as previously described (22) and purified as shown in Fig. 2B, lanes 3 (MacRPA1), 4 (MacRPA2), and 5 (MacRPA3).

The binding site sizes of the MacRPA proteins were determined to be 18 to 23 bases (22). Therefore, as shown in Fig. 3, we tested the effect of each of the three RPA proteins on the capacity of MacFEN1 to cleave a flap 20 nucleotides in length. Interestingly, we observed differences in the effect of the RPA proteins on the endonuclease activity of MacFEN1. As shown in Fig. 3A (i, lane 2), MacFEN1 cleaved the 20-nucleotide flap. However, MacRPA1 inhibited the endonuclease activity of MacFEN1, and addition of this RPA homolog at a concentration of 2.5 pmol/reaction completely inhibited cleavage of the flap (Fig. 3A, i, lane 4). As shown in Fig. 3A (ii), addition of MacRPA2 to the reaction mixture had little influence on the endonuclease activity of MacFEN1. In lane 2, by using a phosphorimager for quantitation, we determined that in the absence of MacRPA2, 64.6% of the substrate was cleaved, whereas addition of 5 pmol, 7.5 pmol, and 10 pmol of MacRPA2 resulted in 60%, 54.4%, and 54.6% of cleaved products, respectively (lanes 5, 6, and 7). MacRPA3 appeared to be more inhibiting to the cleavage of the flap than MacRPA2 (Fig. 3A, iii). In the absence of MacRPA3 (lane 2), 65.7% of the substrate was cleaved. Adding 5 pmol, 7.5 pmol, and 10 pmol of MacRPA3 to the reaction mixture resulted in 50.3%, 40.5%, and 35.0%, respectively, of products (lane 5, 6, and 7). It is interesting to note that our preliminary studies with a double flap substrate, made by adding an extra base (C) to oligonucleotide 2 (5'-CACGTTGACTACCGTCC), also showed that MacRPA1, and not MacRPA2 and MacRPA3, depresses the cleavage of this substrate (see Fig. S1 in the supplemental material).


Figure 3
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  		FIG. 3. Effects of MacRPA proteins on the endonuclease activity of MacFEN1. (A) The effects of MacRPA wild-type proteins on cleavage of a 20-nucleotide flap by MacFEN1. (i) A fixed amount (1 pmol) of a 32P-labeled 20-nucleotide flap substrate (lane 1) was incubated in the presence of MacFEN1 alone (lane 2) and in the presence of MacFEN1 and increasing amounts (1.0, 2.5, 5.0, 7.5, and 10.0 pmol) of MacRPA1 (lane 3, 4, 5, 6, and 7, respectively). (ii) The experiment was the same as described for panel i except that MacRPA2 replaces MacRPA1. (iii) The experiment was the same as in i except that MacRPA3 replaces MacRPA1. (B) The effects of MacRPA wild-type proteins on cleavage of a 5-nucleotide flap by MacFEN1. In panels i, ii, and iii, the experiments were the same as in panel A, except for replacement of the 20-nucleotide flap substrate with that of a 5-nucleotide flap substrate. The products were resolved by 15% denaturing polyacrylamide gel electrophoresis, followed by visualization using autoradiography. The arrows labeled I and II represent cleaved DNA and substrate, respectively.

 
We also tested the effect of MacRPA1 on the cleavage of a flap that was only 5 nucleotides in length. MacFEN1 cleaved the 5-nucleotide flap (Fig. 3B, i), although with less efficiency than in the case of the 20-nucleotide flap, and increasing amounts of MacRPA1 failed to completely inhibit MacFEN1's ability to cleave the short flap. Similarly, MacRPA2 and MacRPA3 did not inhibit or stimulate cleavage of the short flap by MacFEN1 (Fig. 3B, ii and iii).

Eukaryotic RPA proteins have been shown to unwind dsDNA in an ATP-independent manner at low-salt concentrations (26). Since unwinding of the flap substrate by MacRPA1 could affect the activity of MacFEN1, we tested the MacRPA proteins for unwinding of a partial duplex DNA, with a Staphylococcus aureus PcrA helicase as a control. The methanosarcinal RPA proteins did not exhibit an appreciable unwinding activity (see Fig. S2 in the supplemental material). This finding suggested that the inhibitory activity of MacRPA1 on MacFEN1 endonuclease activity was unlikely to be due to unwinding of the substrate by MacRPA1.

The ssDNA binding activities of truncated derivatives of MacRPA1 determined by EMSA. To enhance our understanding of the inhibitory effect of MacRPA1 on MacFEN1 activity, we used deletion analysis of recombinant MacRPA1 to determine which regions of the protein are critical to its inhibition of MacFEN1 endonuclease activity. All truncated mutants were successfully produced and purified from E. coli cells (Fig. 2C). Prior to examining their effects on the endonuclease activity of MacFEN1, we analyzed the ssDNA binding properties of each MacRPA1 derivative to aid us in the interpretation of our results. Three C-terminally truncated mutants of MacRPA1 were made. The first mutant, MacRPA1{Delta}C1, was a MacRPA1 derivative with the last OB fold removed (Fig. 1B, fold D). This mutant clearly bound to ssDNA as shown in the EMSA results in Fig. 4B, although its binding to the substrate appeared to be reduced compared with that of MacRPA1 wild type (Fig. 4A). At lower concentrations of each protein, a single shifted band was prominent. However, as the concentration of each protein was increased, a second and a third slower migrating band were also seen (Fig. 4A and B). In the case of MacRPA1, this observation was not surprising since the estimated binding site size of MacRPA1 is 18 to 23 bases, and the protein is also known to bind to ssDNA as short as 10 bases (22). In the analysis with MacRPA1{Delta}C2, a protein with the last two OB folds removed (Fig. 1B, folds C and D), we could observe only a single shifted band with the same ssDNA substrate and equimolar amounts of proteins as used for MacRPA1{Delta}C1 (Fig. 4C). Deleting three OB folds from the C terminus to yield a MacRPA1 derivative with only one OB fold (MacRPA1{Delta}C3) resulted in a protein lacking detectable ssDNA binding activity in this assay, even at the highest concentration tested (results not shown). Deleting the first and the last OB folds to create a MacRPA1 derivative composed of the two middle OB folds (folds B and C) resulted in a protein (MacRPA1{Delta}N1{Delta}C1) that bound to ssDNA. This truncated derivative exhibited a similar form of protein/ssDNA complex as observed for MacRPA1{Delta}C2 (Fig. 4D), although at 10 pmol of protein per reaction mixture we began to see multiple bands in the EMSA. As shown in Fig. 1B, the three N-terminal truncations led to a protein with three OB folds (MacRPA1{Delta}N1), a protein with two OB folds (MacRPA1{Delta}N2), and finally a derivative with only one OB fold (MacRPA1{Delta}N3). Among the three N-terminally truncated mutants, only MacRPA1{Delta}N1 exhibited ssDNA binding activity in the EMSA (Fig. 4E). Similar to its counterpart with one OB fold deleted from the C terminus, binding of ssDNA by MacRPA1{Delta}N1 was detected at a protein concentration as low as 1 pmol/reaction, although in this case the protein-ssDNA complex was a smear, which may signify unstable binding. As the protein concentration was increased, however, two stable binding states were observed (lanes 4, 5, and 6).


Figure 4
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  		FIG. 4. The ssDNA-binding activities of MacRPA1 and its truncated derivatives by the EMSA method. A fixed amount (2 pmol) of 32P-labeled ssDNA (lane 1) was incubated with increasing amounts (1, 2, 3, 4, and 5 pmol) of each protein under investigation (lane 2, 3, 4, 5, and 6, respectively). Free labeled ssDNA and protein/labeled ssDNA complexes were resolved by 8% PAGE followed by visualization using autoradiography. The proteins under investigation were indicated above each panel. A, MacRPA1 wild type; B, MacRPA1{Delta}C1; C, MacRPA1{Delta}C2, D, MacRPA1{Delta}C1{Delta}N1; and E, MacRPA1{Delta}N1. The arrows labeled I and II represent free labeled ssDNA and protein/labeled ssDNA complex, respectively.

 
The ssDNA-binding activities of MacRPA1 and its truncated derivatives determined by FPA. We applied a different method, FPA, to study the effect of the N- and C-terminal truncations on the capacity of MacRPA1 to bind to ssDNA. This method allowed us to determine the dissociation constant and Hill coefficient, which is an estimate of cooperativity, of each truncated protein that maintained the ssDNA-binding property. All of the C-terminally truncated MacRPA1 derivatives, except for MacRPA1{Delta}C3, were shown by this method to exhibit the ssDNA-binding property as depicted in Fig. 5A. Furthermore, MacRPA1{Delta}C1{Delta}N1 also showed ssDNA-binding activity. These observations were all in agreement with the analysis based on EMSA (Fig. 4B, C, and D). In Fig. 5B, the N-terminally truncated MacRPA1 derivatives were also shown to bind to ssDNA, although similar to the C-terminal truncations, MacRPA1{Delta}N3, a derivative containing only one OB fold, failed to exhibit the ssDNA binding property. By fitting Hill binding curves to the data, we were able to estimate the KD and n values for the proteins that exhibited ssDNA-binding properties (Table 2). Deleting the last OB fold from the C terminus (Table 2, MacRPA1{Delta}C1) did not drastically affect the KD of the protein (5.9 nM versus 5.6 nM of the wild type). In addition, the Hill coefficient (n) was also greater than one (1.4) for MacRPA1{Delta}C1, as also observed for MacRPA1 wild type (Table 2, n = 1.6). In contrast, MacRPA1{Delta}C2 possessed a KD of 22.8, which was about four times higher than the values of the MacRPA1 wild type and MacRPA1{Delta}C1. The Hill coefficient of MacRPA1{Delta}C2 was 1.0, suggesting a protein lacking cooperativity. Although MacRPA1{Delta}C1{Delta}N1 bound to ssDNA, its KD was 16 nM, which was lower than that of MacRPA1{Delta}C2. On the other hand, the Hill coefficient for MacRPA1{Delta}C1{Delta}N1 (1.0) was not different from that of MacRPA1{Delta}C2. Deleting the first OB fold from MacRPA1 resulted in a protein designated MacRPA1{Delta}N1. This truncated derivative possessed a KD of 11.2 nM, which was twice the KD of its counterpart with a single OB fold deleted from the C terminus. Similar to its C-terminally truncated counterpart, the Hill coefficient of MacRPA1{Delta}N1 was greater than one (Table 2, 1.7). Deleting two OB folds from the N-terminal region resulted in a protein that bound to the ssDNA substrate with a KD of ~59 nM. Of the truncated derivatives that maintained the ssDNA binding property, this protein (MacRPA1{Delta}N2) exhibited the lowest affinity for the ssDNA substrate used in the present experiment. Similar to its C-terminally deleted counterpart, deletion of three OB folds from the N terminus resulted in a protein that lacked ssDNA binding activity in the FPA assay.


Figure 5
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  		FIG. 5. Anisotropy measurements reveal binding of ssDNA by MacRPA1 wild type and its truncated derivatives. (A) The binding of MacRPA1 wild type (WT) and its C-terminally truncated products (MacRPA1{Delta}C1 or {Delta}C1, MacRPA1{Delta}C2 or {Delta}C2, MacRPA1{Delta}C3 or {Delta}C3, and MacRPA1{Delta}C1{Delta}N1 or {Delta}C1{Delta}N1) to ssDNA was measured by FPA and fitted to a Hill binding model. (B) The experiments were the same as described in panel A; however, the proteins tested were MacRPA1 wild type (WT) and its N-terminally truncated derivatives (MacRPA1{Delta}N1 or {Delta}N1, MacRPA1{Delta}N2 or {Delta}N2, or MacRPA1{Delta}N3 or {Delta}N3). Each protein was titrated against an 18-base-long ssDNA (FL-18) at a concentration of 15 nM to measure FPA.

 

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  		TABLE 2. Hill coefficients and dissociation constants of MacRPA1 wild type and its derivatives determined by FPAa

 
The specificities of the ssDNA-binding activities of the truncated derivatives of MacRPA1. The truncated derivatives of MacRPA1 that exhibited ssDNA-binding properties in the EMSA were investigated for their ability to discriminate between ssDNA and dsDNA. In Fig. 6A, as we increased the amount of cold ssDNA as challenge in the reaction mixture, binding of MacRPA1 to the labeled ssDNA was out-competed by the cold ssDNA until at 50xexcess cold ssDNA, binding to the labeled ssDNA by MacRPA1 was totally out-competed (lanes 3, 4, and 5). A similar experiment with 10x, 25x, and 50x excess cold dsDNA (lanes 6, 7, and 8) failed to out-compete labeled ssDNA binding by MacRPA1. The binding behavior of MacRPA1{Delta}C1 was not very different from that of the wild-type protein (Fig. 6B), as also shown in Fig. 4A and B, suggesting that the deletion of the last OB fold did not drastically affect the properties of this MacRPA1 derivative. The binding of labeled ssDNA by MacRPA1{Delta}C2, MacRPA1{Delta}C1{Delta}N1, and MacRPA1{Delta}N1 was each out-competed by the levels of excess cold ssDNA tested in the present experiment (Fig. 6C, D, and E, lanes 3, 4, and 5). However, unlike MacRPA1 wild type and MacRPA1{Delta}C1, binding to labeled ssDNA was also out-competed in the presence of 25x, and 50x dsDNA in the reaction mixture (Fig. 6C, D, and E, lanes 7, and 8).


Figure 6
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  		FIG. 6. The specificity of ssDNA binding by the truncated derivatives of MacRPA1 that exhibited binding to this substrate. A fixed amount (1 pmol) of a 32P-labeled ssDNA (lane 1) was incubated with 5 pmol of MacRPA1 or its truncated derivative (lane 2) and challenged with 10 pmol of unlabeled ssDNA (lane 3), 25 pmol of unlabeled ssDNA (lane 4), 50 pmol of unlabeled ssDNA (lane 5), 10 pmol of unlabeled dsDNA (lane 6), 25 pmol of unlabeled dsDNA (lane 7), and 50 pmol of unlabeled dsDNA (lane 8). Free labeled ssDNA and protein/labeled ssDNA complexes were resolved by 8% PAGE followed by visualization using autoradiography. The MacRPA1 truncated derivative under investigation was indicated under each panel. A, MacRPA1 wild type; B, MacRPA1{Delta}C1; C, MacRPA1{Delta}C2; D, MacRPA1{Delta}C1{Delta}N1; and E, MacRPA1{Delta}N1. The arrows labeled I and II represent free labeled ssDNA and protein/labeled ssDNA complex, respectively.

 
The effects of the truncated derivatives of MacRPA1 on cleavage of a 20-nucleotide flap by MacFEN1. As shown in Fig. 7A, deleting one OB fold from the C terminus failed to abolish the inhibitory effect of MacRPA1 on the capacity of MacFEN1 to cleave the 20-nucleotide flap. Note, however, that more of the truncated protein was required to inhibit MacFEN1 activity. At 10 pmol protein per reaction, there was no detectable cleaved product in the reaction with MacRPA1{Delta}C1 (Fig. 7A), whereas in the reaction with MacRPA1{Delta}N1, there was ~10% of cleaved products (Fig. 7B). All other MacRPA1 truncated derivatives lost the inhibitory effect on MacFEN1 endonuclease activity (result not shown).


Figure 7
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  		FIG. 7. The effects of truncated derivatives of MacRPA1 on cleavage of a 20-nucleotide flap substrate. A fixed amount (1 pmol) of a 32P-labeled flap (20-mer) structured DNA substrate (lane 1) was incubated in the presence of MacFEN1 alone (lane 2), and in the presence of MacFEN1 and increasing amounts (1.0, 2.5, 5.0, 7.5, and 10.0 pmol) of the truncated derivative under investigation (lane 3, 4, 5, 6, and 7, respectively). The products were resolved by 15% denaturing PAGE, followed by visualization using autoradiography. The results were designated MacRPA1{Delta}C1 (A) and MacRPA1{Delta}N1 (B). Note that only the results for the MacRPA1 derivatives that maintained the inhibitory activity are shown. The arrows I and II represent cleaved DNA and substrate, respectively.

 
CD. We anticipated that the deletions carried out in MacRPA1 would impact the secondary structural elements or proper folding of the polypeptides. Therefore, CD spectroscopy was used to examine changes in the secondary structure of the truncated mutants. As shown in Fig. 8, the CD spectra of MacRPA1 wild type and its derivatives MacRPA1{Delta}C1 and MacRPA1{Delta}N1 exhibited negative deflections with a single minimum at ~208 nm. The C-terminal truncated derivatives with two (MacRPA1{Delta}C2) and three (MacRPA1{Delta}C3) OB folds deleted also showed negative deflections with a single minimum at approximately 225 nm. However, their mean molar ellipticities were generally different from those of the wild-type MacRPA1 and the truncated derivatives with a single OB fold removed. On the other hand, deleting two (MacRPA1{Delta}N2) and three (MacRPA1{Delta}N3) OB folds from the N terminus resulted in proteins with both negative and positive deflections, and this was strikingly different from the results obtained with the wild-type MacRPA1 protein.


Figure 8
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  		FIG. 8. CD spectra of MacRPA1 wild type (WT) and its truncated derivatives. Triplicate data sets were collected from samples at a concentration of 0.5 mg/ml and normalized against the buffer without protein (50 mM Tris-HCl, pH 8.0, 75 mM NaCl, 0.5 mM DTT). Readings were taken from wavelengths of 200 nm to 260 nm.

 
Distribution of MacRPA1 orthologs. We used the amino acid sequence of MacRPA1 to search the publicly available protein databases for its orthologs. Aside from two other Methanosarcina species (M. mazei and M. barkeri) with completely sequenced genomes, we did not find a protein that matches MacRPA1 in architecture. However, the Halobacterium sp. NRC-1 and Haloarcula marismortui genomes contained genes encoding products that were very similar to the first three OB folds of MacRPA1 and its relatives. It appeared, however, that the halobacterial proteins lack the fourth OB fold (Fig. 9). Our search of the publicly available protein databases suggested that the last 114 amino acids in the halobacterial RPA-like proteins are unique to the two halobacterial proteins. There is also the possibility that this amino acid sequence folds into a noncanonical OB fold. Interestingly, we also found a MacRPA1-like protein in Methanococcoides burtonii. In the polypeptide chain of this protein, we observed a large deletion in the central region (Fig. 9). A careful examination showed that the deletion removed approximately two-thirds of OB fold B from its C terminus and approximately one-third of OB fold C from its N terminus. The result is a new OB fold that is a chimera of OB folds B and C.


Figure 9
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  		FIG. 9. An alignment of MacRPA1, its orthologs in two other Methanosarcina spp., and related MacRPA1-like proteins. The amino acid sequences for the OB folds in MacRPA1 are delineated with the lines between two arrowheads and are labeled with the letters A, B, C, and D. The abbreviations of proteins and their GenBank accession numbers are as follows: MacRPA1, M. acetivorans RPA1 (AAM07979); MmaRPA1, M. mazei RPA1 (NP_633323); MbaRPA1, M. barkeri RPA1 (ZP_00542400); MbuRPA1, M. burtonii RPA-like protein (ZP_00563288); HspRPA1, Halobacterium sp. NRC1 RPA-like protein (NP_279274); and HmaRPA1, H. marismortui RPA-like protein (AAV47126). The conserved and similar amino acids are shaded black and gray, respectively.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we sought to understand whether the conservation of three replication protein A homologs in the Methanosarcinales has led to a "division of labor" among the three proteins. Although we reported previously that each methanosarcinal RPA protein stimulated primer extension by a cognate DNA polymerase (22), the results presented here showed that, compared with MacRPA2 and MacRPA3, MacRPA1 differently affects flap processing by the M. acetivorans FEN1. Three pathways have been proposed for Okazaki fragment maturation in eukaryotes. These are the RNaseH/FEN1, FEN1-only, and Dna2/RPA/FEN1 pathways (15). In the Dna2/RPA/FEN1 pathway, RPA binds a flap of ~27 nucleotides and inhibits cleavage of the flap by FEN1 but not its cleavage by Dna2. Thus, in this pathway RPA binds to the flap and recruits Dna2, which then cleaves most of the flap, leaving a short flap to be processed by FEN1 (1). Through this process, eukaryotic RPA regulates the size of the 5' region removed from the previously synthesized Okazaki fragment, allowing resynthesis of the region by a high-fidelity DNA polymerase. Our demonstration that MacRPA1 inhibits cleavage of the long flap (20 nucleotides) by MacFEN1, and not the short flap (5 nucleotides), suggests that a process similar to the Dna2/RPA/FEN1 pathway may be present in methanosarcinal cells. Further investigations that incorporate other proteins predicted to participate in Okazaki fragment processing, together with genetic analysis, are required to gain a better understanding of the physiological implications of our findings.

The ssDNA binding studies on the truncated derivatives of MacRPA1 yielded interesting insights. Using EMSA, we detected ssDNA-binding activity by MacRPA1{Delta}C2 and not by MacRPA1{Delta}N2. The reason for this difference became evident when we used the biophysical method, FPA, to analyze the ssDNA binding properties of the truncated derivatives of MacRPA1. The KD of MacRPA1{Delta}C2 for ssDNA was 22.8 nM, whereas that of MacRPA1{Delta}N2 for the same ssDNA substrate was about three times this value (59.1 nM). The FPA analysis suggested that neither MacRPA1{Delta}C3 nor MacRPA1{Delta}N3 possessed an ssDNA-binding property. Each of these MacRPA1 derivatives was composed of only one OB fold. Interestingly, deletion of a single OB fold from the C terminus or the N terminus did not severely impair the capacity of these MacRPA1 derivatives to inhibit endonuclease activity of MacFEN1. Thus, it appears that although the C-terminal deletion resulted in a KD of similar value to the wild type and the KD for the N-terminal deletion was about twice that value, neither of the two deletions drastically affected the function or, perhaps, the structure of the protein. These assumptions were supported by the CD analysis (Fig. 8), where MacRPA1 wild type, MacRPA1{Delta}C1, and MacRPA1{Delta}N1 demonstrated very similar CD spectra, whereas the CD spectra of the other truncated derivatives were different. In addition, similar to MacRPA1 wild type, each protein with a deletion of a single OB fold exhibited positive cooperativity (n > 1), suggesting that the binding of ssDNA by each protein increased the affinity of that particular protein for the bound ssDNA during subsequent binding. Note that although the substrate used in the cooperativity study is 18 bases long, which is about the estimated binding site size of MacRPA1, our previous work also showed that MacRPA1 binds well to shorter ssDNAs, such as a 10-base-long ssDNA, albeit with lower affinity. The exact mode (monomer, dimer, or tetramer) by which MacRPA1 binds to ssDNA is not known, and ssDNA binding proteins, such as E. coli SSB, can bind to ssDNA substrates by different modes (20). Thus, a complete explanation for the positive cooperativity shown by MacRPA1 and some of its truncated derivatives may require further investigations. It is also possible that this property, which has been observed even with shorter ssDNA, may be due to protein-protein interactions (22). As stated above, the other truncated derivatives (MacRPA1{Delta}C2, MacRPA1{Delta}C1{Delta}N1, and MacRPA1{Delta}N2) exhibited higher KD values, and in two of the three proteins cooperativity values were also ~1.0, suggesting that each protein binds the ssDNA substrate completely independently of already bound proteins. Thus, MacRPA1 wild type and its truncated derivatives that inhibited MacFEN1 endonuclease activity had at least three OB folds, they exhibited positive cooperativity, and they also possessed better ssDNA binding characteristics. To our knowledge, this inhibitory effect of an ssDNA binding protein on the endonuclease activity of an archaeal FEN1 is being reported for the first time. However, since FEN1 proteins are known to interact with the sliding clamp or proliferating cell nuclear antigen, it may be important in future experiments to determine whether M. acetivorans proliferating cell nuclear antigen modulates the inhibitory effect of MacRPA1 on MacFEN1 endonuclease activity. We have identified a gene encoding a Dna2-like polypeptide (GenBank accession number AAM06856) in M. acetivorans. Although the similarity between the amino acid sequence of biochemically characterized Dna2 and that of the product of the methanosarcinal gene is very low, the M. acetivorans polypeptide shows a reasonable similarity (~35%) across its entire length to an Aspergillus fumigatus putative Dna2 (GenBank accession number XP-751610.1). We plan to express the methanosarcinal gene to determine whether the product exhibits the activities reported for Dna2 proteins. Alternatively, cell extracts can be screened for Dna2 activity with a subsequent search in the M. acetivorans genome for the gene encoding the product with the observed activity. These experiments will enable us to determine whether the Dna2/RPA/FEN1 pathway exists in M. acetivorans cells.

Single-stranded DNA binding proteins are known to prefer ssDNA as a substrate. However, they can also bind to dsDNA, albeit with far lower affinity (26). In fact, it is known that the Saccharomyces cerevisiae RPA can bind certain specific dsDNA with much higher affinity (23). The results shown in Fig. 6, where we challenged the labeled ssDNA binding with excess dsDNA, indicated that in both the wild-type MacRPA1 and MacRPA1{Delta}C1 there was a shift from the third shifted band, which is likely to be bound by more RPA proteins, to the intermediate (faster migrating) band. Thus, in the presence of excess dsDNA, as some RPA proteins dissociated from the labeled ssDNA, they might have bound to dsDNA, leading to fewer MacRPA1 and MacRPA1{Delta}C1 proteins bound to the labeled ssDNA. The results for the other truncated proteins, however, suggested that these deletions resulted in products with less capacity to discriminate ssDNA from dsDNA, and thus less dsDNA was required to out-compete the labeled ssDNA for binding.

In each of three Methanosarcina species with completely sequenced genomes (M. acetivorans, M. mazei, and M. barkeri), the RPA homolog containing four OB folds was present. Our previous analysis showed that the two central OB folds (Fig. 1B, folds B and C) are very similar, sharing identities of ~60% at the amino acid (22) and nucleotide sequence levels. In the psychrotolerant M. burtonii, a member of the Methanosarcinales, we found a variant form of MacRPA1. Based on the alignment in Fig. 9, the M. burtonii putative RPA protein is composed of three OB folds, with the middle OB fold being a chimera of OB folds B and C (Fig. 1B). MacRPA1-like proteins also exist in the salt-loving archaea Halobacterium sp. NRC-1 and H. marismortui. In each organism, however, it seems that the gene has undergone such modifications that only the first three OB folds can be delineated. It is our hypothesis that this unique RPA protein in the Methanosarcinales and the related RPA1-like proteins in the Halobacteriales (Fig. 9) are of common descent. However, the M. burtonii homolog and the halobacterial homolog have evolved to function properly in the ecological setting in which these organisms are found, potentially to adapt to a cold and high-salt environment, respectively. We await, with much interest, the characterization of these interesting proteins in M. burtonii, Halobacterium salinarium, and H. marismortui.


   ACKNOWLEDGMENTS
 
We acknowledge Carl R. Woese for invaluable scientific discussions. We thank William Metcalf (University of Illinois at Urbana-Champaign) for providing M. acetivorans genomic DNA and, together with Roderick I. Mackie (University of Illinois at Urbana-Champaign), for insightful scientific discussions. Tae-Sung Kim and Edgar Hernandez (Cann laboratory) are acknowledged for technical assistance.

This research was supported by a National Science Foundation grant, MCB-0238451, to I.K.O.C. and in part by the Agricultural Experimental Station, University of Illinois at Urbana-Champaign. Also, Y.L. and C.E.G. were supported by National Science Foundation grant MCB-0238451. M.C.M. was supported by Agricultural Genome Sciences and Public Policy training grant 2001-52100-11527 and partially by a National Institutes of Health Institutional NRSA in Molecular Biophysics (5T32GM08276). The research of T.H. is supported by National Institutes of Health grant GM65367.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Animal Sciences, 1207 West Gregory Drive, University of Illinois at Urbana-Champaign, IL 61801. Phone: (217) 333-2090. Fax: (217) 333-8286. E-mail: icann{at}uiuc.edu. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, September 2006, p. 6153-6167, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00045-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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