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Originally published In Press as doi:10.1074/jbc.M504466200 on July 11, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31460-31469, September 9, 2005
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The Protozoan Parasite Cryptosporidium parvum Possesses Two Functionally and Evolutionarily Divergent Replication Protein A Large Subunits*{boxs}

S. Dean Rider, Jr.{ddagger}, Xiaomin Cai{ddagger}, William J. Sullivan, Jr.§, Aaron T. Smith§, Jay Radke¶, Michael White¶, and Guan Zhu{ddagger}||**

From the {ddagger}Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences and the ||Faculty of Genetics Program, Texas A&M University, 4467 TAMU, College Station, Texas 77843, the §Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana 46202, and the Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717

Received for publication, April 25, 2005 , and in revised form, June 24, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Very little is known about protozoan replication protein A (RPA), a heterotrimeric complex critical for DNA replication and repair. We have discovered that in medically and economically important apicomplexan parasites, two unique RPA complexes may exist based on two different types of large subunit RPA1. In this study, we characterized the single-stranded DNA binding features of two distinct types (i.e. short and long) of RPA1 subunits from Cryptosporidium parvum (CpRPA1A and CpRPA1B). These two proteins differ from human RPA1 in their intrinsic single-stranded DNA binding affinity (K) and have significantly lower cooperativity ({omega}). We also identified the RPA2 and RPA3 subunits from C. parvum, the latter of which had yet to be reported to exist in any protozoan. Using fluorescence resonance energy transfer technology and pull-down assays, we confirmed that these two subunits interact with each other and with CpRPA1A and CpRPA1B. This suggests that the heterotrimeric structure of RPA complexes may be universally conserved from lower to higher eukaryotes. Bioinformatic analyses indicate that multiple types of RPA1 are present in the other apicomplexans Plasmodium and Toxoplasma. Apicomplexan RPA1 proteins are phylogenetically more related to plant homologues and probably arose from a single gene duplication event prior to the expansion of the apicomplexan lineage. Differential expression during the life cycle stages in three apicomplexan parasites suggests that the two RPA1 types exercise specialized biological functions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cryptosporidium parvum is a unicellular pathogen that can cause persistent, potentially life-threatening watery diarrhea in both humans and animals. Cryptosporidiosis is especially severe among children and immunocompromised individuals, and there are currently no completely effective therapies available to control cryptosporidiosis (1, 2). This pathogen belongs to the phylum Apicomplexa, which contains many other parasites important to human and animal health (e.g. Toxoplasma, Plasmodium, Babesia, Cyclospora, Isospora, and Eimeria) (3).

All apicomplexans must undergo several different types of cell division to complete their life cycles. One of the intracellular developmental stages is merogony (or schizogony), in which parasite DNA may be replicated up to several hundred times before cytokinesis takes place to simultaneously form several to hundreds of daughter cells (3). This unique cell multiplicative process differs significantly from the typical duplicative cell cycle employed by most other organisms and plays a significant role in apicomplexan pathogenesis, since a large number of parasites can be generated in a single cell cycle. However, although unique and important, little is known about the molecules and mechanisms that govern the DNA replication and cell cycles in apicomplexans.

Replication protein A (RPA)1 is a eukaryotic ssDNA binding complex that plays a central role in DNA replication, repair, and recombination (4–6). In animals, plants, and fungi, RPA is typically a heterotrimeric complex composed of RPA1 (~70-kDa), RPA2 (~34-kDa), and RPA3 (~14-kDa) subunits (5, 6). RPA1 is the major ssDNA binding subunit, and the characterization of this binding has been well documented (5, 6). The RPA2 subunit also has ssDNA binding capacity, but the precise role of the RPA3 protein remains to be elucidated (5, 6). Both RPA1 in Xenopus and RPA2 in yeast are imported into the nucleus in an {alpha}-importin-independent manner (7, 8). For RPA1, the central region containing the major ssDNA binding domains is responsible for interactions with the nuclear import proteins (8). Additionally, the RPA proteins interact with many other proteins involved in DNA metabolism, and regions in the extended N terminus of the RPA1 subunit often mediate these interactions (5, 6). Crystal structures of the human RPA proteins indicate that all three RPA subunits resemble OB folds but that RPA3 lacks key residues implicated in interactions with ssDNA (9–11).

Although RPA complexes from humans and yeast have been well characterized, much less is known about these essential proteins in other organisms. It is of particular interest to study RPAs in apicomplexan parasites, which possess a unique DNA replication mechanism. Among protozoa, only RPA1 subunits from Cryptosporidium parvum (CpRPA1A), Plasmodium falciparum (PfRPA1), and Crithidia fasciculata (CfRPA1) have been examined (12–16). Intriguingly, all three protozoans have been reported to have short forms (~50 kDa) of the RPA1 subunit that lack an extended N-terminal interaction domain (12–16). These observations imply that novel pathways for regulating RPA-mediated DNA metabolism may be present among protists. In addition to the short type CpRPA1A, the C. parvum genome also encodes a second RPA1 subunit (CpRPA1B) of ~70 kDa (16), indicating that two different RPA1 subunits co-exist in apicomplexans. Surprisingly, a smaller (~45-kDa) protein may also be produced from the CpRPA1B locus, but the mechanism regulating this has not been elucidated (16). Recombinant CpRPA1A and CpRPA1B have been generated and shown to bind to ssDNA of different lengths (16). However, the affinity of these unique proteins for ssDNA remains unknown. Among the other RPA subunits (i.e. RPA2 and RPA3), only an RPA2 homologue has been recently identified and characterized from C. parvum (17), and no RPA3 subunit has been reported in any protist to date.

In this study, we have analyzed the ssDNA binding affinities of the two unique C. parvum RPA1 proteins and compared their kinetics with those of human RPA1. Second, we identified and cloned the first protozoon RPA3 subunit from C. parvum (CpRPA3) and validated the interactions between the RPA1, RPA2, and RPA3 subunits using fluorescence resonance energy transfer (FRET) technology and detailed their kinetic parameters. By data mining several parasite genomes, we have also found that different types of RPA1 subunits (i.e. short and long type) are present in at least three major apicomplexan lineages. The two different types of apicomplexan RPA1 subunits are differentially expressed among parasite life cycle stages, suggesting that they may play different biological roles in the complex apicomplexan life and cell cycles. Our data also indicate that conserved differences between the apicomplexan RPA1 subunits and the human RPA1 may facilitate the study of these proteins as potential drug targets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Constructs—Constructs for expressing CpRPA1A, CpRPA1B, and CpRPA2 as maltose-binding protein (MBP) fusions were described previously (16, 17). To facilitate direct comparison of biochemical features between parasite and human RPA1 subunits, a construct for expression of the human RPA1 (HsRPA1) as an MBP fusion was also generated. The HsRPA1 open reading frame (ORF) was PCR-amplified from an OmicsLink expression vector (catalog number EX-B0001-B01; GeneCopoeia) with Pfu Turbo (Stratagene) using the following primers: 5'-AAC gaa ttc ATG GTC GGC CAA CTG AG-3' and 5'-AAC gga tcc TCA CAT CAA TGC ACT TC-3' (lowercase indicates engineered restriction sites for cloning). The amplified product was purified from an agarose gel slice using a Min Elute kit (Qiagen) and cloned into the pCR4Blunt-TOPO vector according to the manufacturer's instructions (Invitrogen). The HsRPA1 sequence was verified, and the gene was cloned into the pMAL-p2X vector (New England Biolabs) using EcoRI and BamHI. A construct for expressing an MBP fusion of CpRPA1B lacking an N-terminal extension (CpRPA1B{Delta}) was also generated using similar methods. The primers used were 5'-Cg aat tcA TGG GTT CAG GTC AGT CAA AG-3' and 5'-Cga att cTC AAT ATC CAA ACT GTC CAA ATC-3'. The putative CpRPA3 gene was identified from the C. parvum genome by repeated BLAST searches using various known RPA3 proteins as queries. The candidate locus was then amplified by PCR from parasite genomic DNA or by RT-PCR from total RNA using a OneStep RT-PCR kit (Qiagen). Both genomic DNA and RNA were isolated from purified C. parvum sporozoites as previously described (18). The following primers were used: 5'-ATG CAG AGC TCA ATT GAA AAT G-3' and 5'-CTA AGC AAT ATT AGA AAC AGG CT-3'. The product generated from cDNA was smaller than the product generated from genomic DNA, confirming the presence of an intron that was predicted to be present within the genomic locus. Therefore, the CpRPA3 cDNA was cloned into the pCR2.1 vector (Invitrogen) as described for the HsRPA1 locus. Following confirmation of the sequence, the CpRPA3 ORF was engineered into the pMAL-p2X vector at XmnI/EcoRI restriction sites without an ORF shift.

The CpRPA3 ORF was also amplified with the following primers: 5'-CGg gat ccA TGC AGA GCT CAA TTG AAA ATG-3' and 5'-GCg aat tcT AAG CAA TAT TAG AAA CAG GCT-3' and engineered into the pET-24a(+) vector (Novagen) at the BamHI and EcoRI sites. A 0.5-kb XbaI/HindIII fragment from this vector was then transferred to the MBP-CpRPA2 vector mentioned above. A 1.4-kb XmnI/HindIII fragment was then transferred into the pET29a(+) vector (Novagen) at the EcoRV/HindIII sites. This construct was capable of expressing S-tagged CpRPA2 and T7-tagged CpRPA3. A 1.5-kb fragment from this construct was then transferred into three other vectors. The first two vectors were engineered with the pMAL-p2X vector or the MBP-CpRPA1A expression vectors using the XbaI and HindIII restriction sites. Similarly, the third vector was engineered with the MBP-CpRPA1B expression vector using XbaI and SalI. These three new vectors were designed to express S-CpRPA2 and T7-CpRPA3 along with MBP, MBP-CpRPA1A, or MBP-CpRPA1B.

All expression constructs were sequenced to confirm the orientation and sequence of cloned fragments. Each construct was maintained and amplified in Escherichia coli MachI cells (Invitrogen) grown in the presence of glucose (2 mM) to repress expression of the fusion proteins.

Protein Expression and Purification—Plasmid DNA from the expression vectors described above was isolated from the MachI host cells using a QIAprep mini spin kit (Qiagen) and transferred into E. coli Rosetta cells (Novagen). Transformed Rosetta cells were plated and grown overnight at 37 °C on solid LB medium containing chloramphenicol (34 µg/ml), ampicillin (50 µg/ml), and glucose (2 mM). Conditions for expressing MBP-fused parasite and human RPA proteins were similar to those described previously (16, 17). Briefly, six clones were individually inoculated into 5 ml of liquid LB (with chloramphenicol, ampicillin, and glucose), and cultures were grown overnight at 30 °C. The following morning, all six cultures were combined and transferred into a 2-liter flask containing 1 liter of LB medium (with antibiotics or glucose) and grown for an additional 5 h. Isopropyl 1-thio-{beta}-D-galactopyranoside was added to the culture, and the cells were allowed to grow for an additional 2.5 h prior to harvesting. Harvested cells were resuspended in 40 ml of TNE buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, and 1 mM EDTA). Cells were lysed by mild sonication and centrifuged to remove insoluble debris, and the MBP fusion proteins were then purified using an amylose resin-based affinity chromatography (New England Biolabs) (16, 17). Purified proteins were dialyzed extensively against 1x Dulbecco's PBS (Sigma) before use.

Electrophoretic Mobility Shift Assays—Oligonucleotides (dT20 or dT50) were end-labeled using [{gamma}-33P]ATP (PerkinElmer Life Sciences) and T4 polynucleotide kinase (New England Biolabs) according to the manufacturer's instructions. Unincorporated nucleotides were separated from oligonucleotides by chromatography through G50 macro spin columns (Harvard Biosciences), and aliquots of labeled oligonucleotides were diluted to concentrations that ranged from 1 to 400 nM. ssDNA binding reactions were performed using an excess of recombinant MBP-RPA1 protein (~1 µgin10 µlof1x Dulbecco's PBS) and 5 µl of diluted oligonucleotide. The final buffer composition of the binding reactions was 0.5x PBS in 20 mM Tris-HCl, pH 7.4. After a 30-min incubation at 25 °C, bound oligonucleotides were cross-linked to the RPA protein by exposing open reaction tubes to 120 mJ/cm2 ultraviolet light using a Stratalinker 1800 (Stratagene). UV levels beyond 100–120 mJ/cm2 did not result in an increase in cross-linked products, indicating that the cross-linking was near maximal under these conditions (data not shown). Cross-linked products were mixed with an equal volume of 2x sample buffer (20 µl), heated to 65 °C for 10 min, and subjected to SDS-PAGE (19). A Bio-Rad Mini-PROTEAN gel system was used for casting and running continuous 1.5-mm-thick 6% polyacrylamide gels. Gels were cast in the presence of GelBond PAG film (Amersham Biosciences) to provide a solid support for and to prevent distortion of the gels following electrophoresis. Electrophoresis was conducted in Tris/glycine buffer containing SDS (2.5 mM Tris, 19 mM glycine, 0.1% SDS). Gels were subjected to current at 20 mA for 30 min prior to the loading of samples, which were also electrophoresed for 30 min at 20 mA. Following electrophoresis, gels were rinsed briefly with running buffer, wrapped with plastic film, and exposed to an imaging plate (BAS MS 2345; FujiFilm) for several hours. Gel images were collected using a BAS-1800 II Bio Imaging Analyzer (Fujifilm), and image analysis was performed using NIH Image (available on the World Wide Web at rsb.info.nih.gov/nih-image/Default.html). Fractional binding data were analyzed as described previously (20, 21) using nonlinear regression and GraphPad Prism 3.0 software.

Fluorescence Labeling of Recombinant CpRPA Proteins—MBP-fused parasite proteins were directly labeled with appropriate fluorescent probes for FRET analysis. Prior to labeling, the concentrations of CpRPA1A and CpRPA1B were adjusted to 0.11 mg/ml, whereas those of CpRPA2 and CpRPA3 were at 2 mg/ml in 1x Dulbecco's PBS. Recombinant proteins were labeled with either Alexa Fluor 488 (excitation = 495 nm, emission = 519 nm) or Alexa Fluor 546 (excitation = 556 nm, emission = 573 nm) labeling kits following the recommendations of the manufacturer (Molecular Probes, Inc., Eugene, OR) and included an overnight incubation at 4 °C prior to column purification of the labeled proteins. These fluorophores represent a fluorescence resonant energy transfer (FRET) pair with a Forster radius (Ro) of ~64 Å. CpRPA1A, CpRPA1B, and CpRPA2 were all labeled with the donor fluorophore (Alexa Fluor 488), whereas CpRPA2 and CpRPA3 were labeled with the acceptor fluorophore (Alexa Fluor 546). A portion of labeled MBP-CpRPA2 was also digested with Factor X{alpha} protease (Novagen) to cleave the MBP domain from the CpRPA2. The MBP was removed by incubating the digestion reactions with an excess of amylose resin that had been equilibrated with 1x Dulbecco's PBS, the liquid fraction was collected, and an aliquot was analyzed by SDS-PAGE to confirm the cleavage and removal of the MBP tag. The amylose resin with bound, labeled MBP was subsequently incubated with 1x Dulbecco's PBS containing maltose to elute the fluorophore-labeled MBP.

Protein Interaction Assays—To determine whether any of the CpRPA proteins may interact with each other, we quantified the ability of an Alexa Fluor 546-labeled CpRPA subunit to quench the fluorescence of another Alexa Fluor 488-labeled CpRPA subunit. Interactions between pairs of CpRPA subunits were examined, which include MBP-CpRPA1A with MBP-CpRPA2, CpRPA2, or MBP; MBP-CpRPA1B with MBP-CpRPA2, CpRPA2, or MBP; MBP-CpRPA2 with MBP-CpRPA3; MBP-CpRPA1A with MBP-CpRPA3; and MBP-CpRPA1B with MBP-CpRPA3, respectively. Reactions were set up in 96-well, autofluorescence-free black plates (catalog number 9502867; Thermoelectron Corp.) and were performed in the binding buffer described above for ssDNA binding reactions. In these experiments, a fixed amount of donor labeled protein was incubated with increasing amounts of acceptor labeled protein. Following the addition of acceptor, fluorescence measurements of all reactions were performed as described below.

Fluorometry—All fluorescence measurements were performed using a Fluoroskan Ascent fluorometer (Thermoelectron). The fluorometer was programmed to maintain a constant temperature of 25 °C and to shake samples for 20 s at 120 rpm (1-mm diameter rotation) prior to measuring the fluorescence of the samples (20–60-ms integration time, normal beam). The fluorometer was fitted with two different sets of band pass interference excitation and emission filters: one for Alexa Fluor 488 (excitation = 485/7 nm; emission = 538/12.5 nm) and one for Alexa Fluor 546 (excitation = 544/7.5 nm; emission = 590/7 nm). FRET was also measured using the mixed filter set (excitation = 485/7 nm and emission = 590/7 nm). However, we found that the signal obtained from Alexa Fluor 488 using this filter set was a significant source of background that made measurements of FRET difficult. Therefore, interactions were based upon the ability of acceptor-labeled proteins to quench the fluorescence of donor-labeled proteins. Fluorescence measurements of individual reactions were based on the average of 3–5 scans, and each experiment was replicated three times. Control assays included separate reactions with donor or with acceptor as a means of monitoring photobleaching and absorbance artifacts that may have occurred during the experiment and for normalizing the data.

Protein Pull-down Assays—Whole-cell lysates from E. coli engineered to co-express S-CpRPA2 and T7-CpRPA3 along with MBP, MBP-CpRPA1A, or MBP-CpRPA1B were generated as described under "Protein Expression and Purification." A portion of each lysate was reserved for examining the expression of the proteins of interest (input). Amylose resin (0.5 ml) that had been equilibrated with TNE buffer was added to the remainder of each lysate. After a 30-min incubation (25 °C), the resin was collected by centrifugation (pull-down) and rinsed once with 20 ml of TNE buffer. Proteins bound to the amylose resin were released by incubating the resin in 0.75 ml of TNE containing maltose. Proteins from the lysate and the eluted pull-down fractions were quantified using the Bio-Rad protein assay and subjected to Western or slot blot analysis. Alkaline phosphatase-conjugated S-protein (Novagen) or anti-T7 antibody (Novagen) was used as a basis for detecting S-CpRPA2 or T7-CpRPA3, respectively. Alkaline phosphatase was detected using SigmaFast (Sigma).

Transcript Analysis for Apicomplexan RPA Large Subunits at Various Developmental Stages—For C. parvum (IOWA strain), oocysts freshly isolated from infected calves were purchased from Bunch Grass Farm (Troy, ID), further purified by Percoll gradient centrifugation, and stored in water at 4 °C at Texas A & M University (22). Free sporozoites were prepared by excystation of oocysts in PBS containing 0.1% trypsin and 0.5% taurodeoxycholic acid for 90 min at 37 °C and purified by Percoll gradient centrifugation as described previously (23). Intracellular stages were obtained by infecting human HCT-8 cells with C. parvum oocysts for various times (i.e. 12–72 h) and harvested at every 12 h postinfection for isolating RNA. Total RNA was isolated from various C. parvum developmental stages using a Qiagen RNAeasy kit according to the manufacturer's recommendations for animal cells. For oocysts, 5–10 freeze-thaw cycles (liquid nitrogen and 37 °C) were used to disrupt the oocyst cell wall prior to RNA isolation (24).

Total RNA was isolated from partially sporulated VEG strain oocysts that were obtained by sucrose flotation from cat feces as previously described (25). Briefly, an ~10-ml volume of solid fecal material was vortexed with an equal volume 1.1 M sucrose solution, over which 10 ml of deionized water was then layered. The gradient was then centrifuged for 20 min at 115 x g, the water layer and interface (not sucrose) were removed to a fresh conical tube, and the process was repeated on the remaining slurry. The two extracts were combined, and water was added to a final volume of 50 ml. The oocysts were pelleted from the extract by centrifugation, resuspended in 10 ml of 2% sulfuric acid, and allowed to sporulate for 2–3 days at room temperature (full sporulation requires 5 days). Oocysts were pelleted and resuspended and incubated for 30 min at room temperature in 10% (v/v) Clorox (in PBS), collected by centrifugation again, and washed four times in Hanks' balanced salt solution (Gibco). Partially sporulated oocysts (30 x 106) were resuspended in 4 ml of 4 M guanidine thiocyanate solution (20 mM sodium acetate and 0.5% N-laurylsarcosine), transferred to a French pressure cell (catalog number FA003, 20,000 p.s.i.; Sims-Aminco by Thermo Electron Corp., Needham Heights, MA), and frozen for 2 min at –80 °C. Frozen oocysts were immediately crushed at 20,000 p.s.i. in a French pressure cell press (model FA-078; Sims-Aminco by Thermo Electron). Crushed oocysts were extracted two times with an equal volume of chloroform according to standard methods, and RNA was precipitated by standard methods and resuspended in injection grade water.

Toxoplasma gondii ME49 strain was grown in human foreskin fibroblasts using Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum. Tachyzoites were grown at 37 °C in 5% CO2 and harvested following egress from the host cell monolayer. For bradyzoite samples, parasites were induced to differentiate 4 h postinfection by applying CO2 starvation medium (26) and incubating at 37 °C in ambient CO2 for 7 days. Any extracellular parasites were removed daily by aspiration and replacement of culture medium. Bradyzoite cyst formation was confirmed by microscopy, and bradyzoites were harvested by physically disrupting the host cell monolayer and cyst wall. Parasite mRNA from either stage was isolated from filter-purified parasites using the Poly(A)-Pure system (Ambion). To further confirm the fidelity of the in vitro differentiation and purity of the tachyzoite fraction, RT-PCR using stage-specific primers was employed (data not shown).

The transcript levels of C. parvum and T. gondii RPA1 subunits at various developmental stages were determined by SYBR-green-based real time quantitative RT-PCR using an iCycler iQ system (Bio-Rad) (27). Primers used in the quantitative RT-PCR are summarized in Table I. The levels of C. parvum 18 S rRNA and T. gondii cytosolic glyceraldehyde-3-phosphate dehydrogenase (TgGAPDHc) mRNA were also determined for standardization controls. Each reaction contained at least three replicates. Standard curves for all primer pairs were determined using serially diluted DNA templates in real time PCR analysis, followed by linear regression to determine needed parameters (i.e. curve slopes and intersections). Based on the standard curves, the transcript level for each C. parvum and T. gondii gene was determined. The relative levels of C. parvum and T. gondii RPA1 transcripts were expressed relative to those of 18 S rRNA and TgGAPDHc, respectively.


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  		TABLE I
Primers used for gene expression analysis with real time PCR

 



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  		FIG. 1.
Saturation ssDNA binding curves for MBP fusions of the RPA1 proteins from human (HsRPA1) and Cryptosporidium parvum (CpRPA1A, CpRPA1B, and CpRPA1B{Delta}). Binding curves are based on electrophoretic mobility shift assays with increasing amounts of oligo(dT50) or oligo(dT)20. The data represent the average of three experiments along with the S.D. value. The inset of each curve shows a representative gel for each experiment (B, bound oligo; F, free oligonucleotide). The estimated macroscopic binding constants (Kd) are presented.

 
We also examined the expression levels of P. falciparum RPA1 subunits by mining the raw expression data from a previously published microarray data base deposited on the World Wide Web at plasmodb. org (28). The Affymetrix-based raw data were analyzed and summarized for assessing the expression levels of P. falciparum RPA1 subunits.

Phylogenetic Reconstructions—To test whether the two types of apicomplexan RPA1 subunits shared the same evolutionary ancestor, we also performed maximum likelihood (ML)-based phylogenetic analyses. RPA1 orthologues in apicomplexans, animals, plants, and fungi were identified by repeated PSI-BLAST searches against various genome data bases using known RPA1 sequences as queries. New apicomplexan putative RPA sequences were retrieved from the following genome data bases: cryptodb.org (C. parvum); toxodb.org (T. gondii; also see supplemental information); and plasmodb.org (P. falciparum). The remaining sequences were retrieved from GenBankTM.

RPA1 protein multiple sequence alignments were performed using the ClustalW algorithm. Apparent mistakes in the alignment were corrected based upon visual inspection. Only amino acid positions that could be unambiguously aligned were selected for phylogenetic analyses. ML trees were constructed using the PROML program distributed in the PHYLIP package (evolution.gs.washington.edu/phylip.html). The JTT model of amino acid substitutions (29) was used in ML analysis with the consideration of among-site heterogeneity using the fraction of invariance plus a four-rate {Gamma}-distribution model (JTT-f + {Gamma} + Inv, where JTT-f means Jones-Taylor-Thornton-f and Inv means Invariant). Sequence input orders were randomized with 10 jumps, and global rearrangements were enabled during the tree searches. Missing parameters required by the PROML program were estimated by the Tree-Puzzle version 5.2 program (30). In addition, ML trees were also reconstructed using a Bayesian inference (BI) method using the same model of amino acid substitutions (i.e. JTT-f +{Gamma}+ Inv) (31). A total of 500,000 generations of searches were performed with four chains simultaneously running, and the current trees were saved every 100 generations. Posterior probabilities at tree nodes were obtained by calculating consensus trees from the BI trees written after the ML sums converged.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The ssDNA Binding of CpRPA1A and CpRPA1B Differs from That of Human RPA1—We have previously identified two different types of RPA1 subunits from C. parvum (i.e. short type CpRPA1A and long type CpRPA1B). Using recombinant proteins in ssDNA-binding studies, CpRPA1A was found to bind oligonucleotides as short as 20 nucleotides, whereas CpRPA1B was shown to bind oligonucleotides as short as 5 nucleotides (16). In contrast, the human RPA1 has been reported to bind oligonucleotides of 8 nucleotides or longer (32). However, detailed ssDNA-binding kinetics of CpRPA1A and CpRPA1B were not established.

To determine the ssDNA binding affinity of CpRPA1A and CpRPA1B in comparison with that of the human RPA1 (HsRPA1), we used electrophoretic gel mobility shift assays along with the MBP fusion proteins that were expressed and purified from E. coli (Fig. 1). The possibility of the CpRPA1B locus producing a peptide lacking the N-terminal domain was intriguing; therefore, we also examined the ssDNA binding ability of an N-terminal deletion of this protein (CpRPA1B{Delta}). Both MBP-HsRPA1 and MBP-CpRPA1A produced two major bands with an oligo(dT)50, indicating the presence of oligonucleotides bound by one or two RPA1 proteins. In contrast, both MBP-CpRPA1B and MBP-CpRPA1B{Delta} produced three major bands, indicating 1–3 RPA1 proteins/oligonucleotide. Additional faster migrating bands were observed with both MBP-HsRPA1 and MBP-CpRPA1B (both proteins contain extended N termini) that were not observed in reactions where the products were not cross-linked to DNA (16, 33). In all cases, the bands corresponding oligonucleotides bound with multiple RPA1 proteins were less intense than the singly bound oligonucleotides, indicating that the RPA1 fusion proteins were not being depleted in these binding reactions. Data derived from binding reactions that contained increasing amounts of radiolabeled oligonucleotides yielded saturable binding curves for all RPA1 fusion proteins. The estimated macroscopic dissociation constants for each protein were slightly different. However, all proteins bound an oligo(dT)50 with picomolar affinity. Moreover, CpRPA1B possessed the same affinity for the oligo(dT)50 regardless of the presence or absence of the N-terminal extension.

The ability of both the HsRPA1 and CpRPA1A proteins to bind twice to the oligo(dT)50 allowed us to estimate the intrinsic binding (K), as well as the level of cooperativity ({omega}). However, to obtain data whereby CpRPA1B was maximally bound only twice to an oligonucleotide, a smaller oligo(dT20) was utilized. A two-site binding model was used to estimate both the microscopic binding and cooperativity (21). The model that best fit the data indicated that the intrinsic ssDNA binding for all three RPA1 proteins was in the nanomolar range (Table II). Whereas the CpRPA1A protein appears to have a somewhat weaker binding than the human RPA1, the CpRPA1B protein appears to have much stronger affinity for ssDNA. The models used indicated that all three proteins had low cooperativity (Table II). Thus, it appears that the CpRPA1 proteins do not promote the binding of additional homologous RPA1 proteins to ssDNA. The values obtained for the MBP-HsRPA1 are consistent with previously published data (nanomolar binding affinity and cooperativity of ~1.8) (20). The observed differential ssDNA binding features (K and {omega}) between human and C. parvum RPA1 proteins and between the two types of parasite RPA1 subunits are consistent with the previously published data on HsRPA1 and on minimum lengths of oligonucleotides required for CpRPA1A and CpRPA1B binding (16, 20). Interestingly, values for {omega} for both C. parvum RPA1 proteins were ~5-fold smaller than that estimated for the human protein. Thus, the Cryptosporidium RPA1 proteins differ from human RPA1 with respect to the length of oligonucleotides that can be bound as well as the intrinsic binding and cooperativity.


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  		TABLE II
Binding parameters of Human and Cryptosporidium RPA1 proteins to ssDNA

A two-site binding model was used to fit data from saturation binding experiments in Fig. 1. The microscopic binding constant (K) and cooperativity ({omega}) values were estimated from the data. The MBP-HsRPA1 fusion was included for comparison and provides estimates similar to those previously reported for the human RPA. Both CpRPA1A and CpRPA1B are estimated to have very low cooperativity values. All three proteins have high intrinsic ssDNA binding affinity.

 
CpRPA2 Has Affinity for both CpRPA1A and CpRPA1B— The RPA heterotrimer is conserved from yeast to insects to humans. The presence of both long and short RPA1 subunits in C. parvum and only one RPA2 subunit contrasts with what is seen in humans. This raised the intriguing question of whether the two different types of C. parvum RPA1 subunits can form heterotrimeric complexes with the same set of RPA2 and RPA3 subunits. One effective approach to address this question is to test whether either CpRPA1A or CpRPA1B or both may interact with CpRPA2. This would also confirm that the putative CpRPA2 protein is indeed one of the RPA subunits. We employed FRET technology, which would allow the detection of protein-protein interactions under mild physiological conditions. In these experiments, MBP-CpRPA1A and MBP-CpRPA1B were labeled with Alexa Fluor 488, whereas MBP-CpRPA2 was labeled with Alexa Fluor 546. A preliminary experiment demonstrated that fluorescence-labeled RPA1 proteins maintained their ability to bind ssDNA (data not shown). Alexa Fluor 488 and 546 represent a FRET pair, in which Alexa Fluor 488 is the donor fluorophore and Alexa Fluor 546 is the acceptor. The Forster radius (Ro) for this FRET pair is ~64 Å, suggesting that energy transfer may occur only when two proteins are present in a close proximity (i.e. 0–128 Å), a distance that can be maintained when two proteins are indeed interacting with each other.



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  		FIG. 2.
Binding reactions indicating CpRPA1A and CpRPA1B interaction with CpRPA2. The labels above each graph indicate which donor (listed first) and acceptor (listed second)-labeled proteins were examined, with carats indicating the direction of energy transfer. The ordinate axis scale on the left indicates the relative fluorescence (%Fo = percentage of initial fluorescence) of donor-labeled protein for control reactions (open circles) or reactions with increasing amounts of acceptor-labeled protein (shaded circles). The ordinate axis scale on the right indicates the relative fluorescence (%Fmax = percentage of maximum fluorescence) of the acceptor for control reactions (open squares) or acceptor-labeled protein in the presence of donor-labeled protein (shaded diamonds). Concentrations of acceptor-labeled protein are indicated by the x axis. Significant quenching of donor-labeled CpRPA1A or CpRPA1B protein is observed in the presence of acceptor-labeled MBP-CpRPA2, or CpRPA2 protein, indicating a protein-protein interaction. The acceptor-labeled proteins have the same expected linear increase in fluorescence in the presence or absence of donor. Control reactions with increasing amounts of acceptor-labeled MBP displayed variable levels of quenching that roughly approximated a linear decrease that is indicative of nonspecific binding (data not shown).

 
By measuring the quenching of emission from the donor at 538 nm (excited at 485 nm), we found that increasing the amount of MBP-CpRPA2 in binding reactions with either MBP-CpRPA1A or MBP-CpRPA1B resulted in saturable quenching curves with both MBP-CpRPA1A and MBP-CpRPA1B (Fig. 2). In binding reactions, as well as control groups lacking CpRPA1A or CpRPA1B, increasing amounts of added acceptor-labeled protein resulted in the expected linear increase in emission for the acceptor protein at 590 nm (excited at 544 nm) (Fig. 2). This observation indicated that, at the concentrations used in these experiments, primary absorbance artifacts (also known as inner filter effects) were not the cause of the quenching observed for the donor in the binding reactions. The apparent dissociation constants for these interactions were in the nanomolar range (Table III). To determine whether the quenching was affected by the presence of the MBP tag, we performed similar reactions using either the MBP tag alone or with tagless CpRPA2. Binding experiments with the MBP portion resulted in slight quenching that was highly variable and did not follow a sigmoidal or hyperbolic dose-response curve (data not shown), indicating that low levels of nonspecific interactions between MBP and RPA subunit might occur, but at levels without interfering with the specific interactions between RPA subunits. On the other hand, FRET experiments with tagless CpRPA2 resulted in saturable quenching curves for both MBP-CpRPA1A and MBP-CpRPA1B. Only slightly lower apparent dissociation constants were observed with tagless CpRPA2 compared with MBP-tagged CpRPA2 (Table III). These observations confirm that CpRPA2 was responsible for the observed quenching and imply that CpRPA2 can interact with either CpRPA1A or CpRPA1B.


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  		TABLE III
Dissociation constants for interactions between various C. parvum RPA subunits as determined by FRET assays

 
Cryptosporidium Possesses a Highly Divergent RPA3 That Is Able to Interact with the Other RPA Subunits—Because both RPA1 and RPA2 subunits had been identified in C. parvum, and the RPA complex is typically present as a heterotrimer, we were interested in identifying any putative RPA3 subunits that may be present in the C. parvum genome. Previously, no homologues of the RPA3 subunit had been studied in any protozoa. An examination of the completed C. parvum genome using BLASTx searches revealed a single, previously annotated ORF at locus cgd3_3400 with similarity to the RPA3 consensus. However, the ORF lacked a start codon, suggesting that this locus may be a pseudogene or may be interrupted by introns. Further examination of the ORF and surrounding genomic DNA using GENSCAN (available on the World Wide Web at genes.mit.edu/GENSCAN.html) predicted that the putative RPA3 locus contained an intron. This was an intriguing result, given that only about 5% of C. parvum loci appear to contain introns (34). We confirmed the presence of this intron by amplifying the putative RPA3 locus from both cDNA and genomic DNA and comparing the products by both gel electrophoresis and DNA sequencing (Fig. 3). An alignment of the predicted C. parvum RPA3 with other known or putative RPA3 proteins indicated limited similarity among the RPA3 proteins (Fig. 3).

To test whether this locus encoded a bona fide homologue of RPA3 (CpRPA3), we expressed the ORF as an MBP fusion protein and examined its ability to interact with recombinant CpRPA1A, CpRPA1B, and CpRPA2 using FRET technology. We observed a saturable quenching curve for each protein pair (Fig. 4), indicating that CpRPA3 was able to specifically interact with the other CpRPA subunits with apparent Kd values ranging from 4.2 to 6.5 nM (Table III).

We also examined whether CpRPA2 and CpRPA3 interact with both CpRPA1A and CpRPA1B using a pull-down assay with lysates from cells that co-expressed CpRPA2 as an S-tagged protein along with T7-tagged CpRPA3 and one of the following: MBP, MBP-CpRPA1A, or MBP-CpRPA1B (Fig. 5). S-CpRPA2 and T7-CpRPA3 were expressed in all three samples as indicated by the signals obtained from the input samples. Both S-CpRPA2 and T7-CpRPA3 have high affinity for CpRPA1A and CpRPA1B as indicated by the presence of a strong signal obtained from those pull-down samples. These observations corroborate the protein interactions indicated by the FRET assays and suggest that the RPA heterotrimeric complex is conserved in C. parvum.

Different Types of RPA1 Subunits Are Commonly Present among Apicomplexan Parasites and Probably Evolved from a Common Ancestor—The presence of two different types of RPA1 in C. parvum raises a question of whether this is common among apicomplexans. To address this question, we searched the P. falciparum and T. gondii genomes (available on the World Wide Web at plasmodb.org and toxodb.org, respectively) for RPA orthologues. Like C. parvum, the P. falciparum genome is predicted to encode two RPA1 subunits, a single RPA2, and a single RPA3 subunit (Table IV). The two RPA1 subunits from P. falciparum contain ORFs predicted to encode a long (~134 kDa, PFD0475c) and a short (~56 kDa, PFI0235w) RPA1 protein. The unusual transcript of PFD0475c has been previously reported to unexpectedly encode a 50-kDa protein, although its ORF predicts a 134-kDa protein (14). The N-terminal extension was not present in native proteins isolated from P. falciparum using ssDNA-coupled affinity chromatography. However, a peptide fragment corresponding to the PFD0475c N-terminal extension was detected from P. falciparum gametocytes by a mass spectrometry-based proteomic analysis (see PFD0475c entry on the World Wide Web at plasmodb.org, under proteomics data). This implies that two types of proteins may be produced from this long type PFD0475c transcript. It also agrees with our previous observation that two protein bands are recognized by antibodies specific to the long type CpRPA1B protein (16).


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  		TABLE IV
Apicomplexan homologues of RPA subunits

The predicted proteins are listed along with the corresponding data base ID (following the nomenclature of the respective genome consortiums). A reference to the protein, if previously studied, is given along with the gi number from the public databases (when available). The predicted size of the protein, based upon the conceptual translation, is also presented. Cp, C. parvum; Pf, P. falciparum; Tg, T. gondii. The predicted protein sequences for T. gondii RPA subunits are given as supplementary materials. All other sequences are available from GenBankTM.

 
In contrast, the T. gondii genome appears to encode three RPA1 subunits, one RPA2 subunit, and an RPA3 subunit (Table IV). Additionally, one other predicted protein (TgTwin-Scan_5993) shows weak similarity to the RPA3 proteins. The predicted RPA1 proteins from T. gondii include a long form (~98 kDa, locus TGG_994685), an intermediate form (58.8 kDa, TGG_994642), and a short form (57.5 kDa, TGG_994459). These observations indicate that the presence of multiple RPA1 subunits is not unique to the Cryptosporidium lineage but is conserved among apicomplexans.

Both long and short forms contained conserved regions corresponding to the major ssDNA binding domains and nuclear import region of the RPA1 proteins. Further sequence analyses validate that all longer forms of apicomplexan RPA1 proteins (>70 kDa) have a domain structure similar to that of the canonical (human and yeast) RPA1 proteins, including a highly divergent N-terminal region and a C-terminal zinc finger motif (Fig. 6), whereas the short type apicomplexan RPA1 proteins lack an N-terminal extension. A search for conserved domains indicated that the long forms, but not the short forms, contained a tRNA anticodon-like OB fold motif within the ssDNA binding domains. Multiple sequence alignments of the ssDNA binding domains of the apicomplexan and human RPA1 proteins indicated that the region containing the tRNA anticodon-like OB fold was well conserved among the apicomplexan long forms but that this region was divergent and contained small deletions in the apicomplexan short forms (Fig. 6). Moreover, this analysis suggested that TGG_994642-encoded T. gondii RPA1 might represent a highly divergent (perhaps intermediate) member within the apicomplexan RPA1 family. Interestingly, the region surrounding the deletion that is present in the apicomplexan short forms corresponds to the area of the human RPA1 that interacts with RAD51 and RAD52, proteins involved in homologous recombination and DNA repair (35, 36). The observed dichotomy between the apicomplexan RPA proteins suggests that the two forms may have different protein regulators or perform different functions.



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  		FIG. 3.
Analysis of the CpRPA3 locus. The conceptual translation of the sequenced CpRPA3 cDNA is presented aligned with putative RPA3 proteins from apicomplexans and the RPA3 proteins from yeasts and humans. Shaded amino acids represent 50% or greater conservation. Numbers to the right of the alignment indicate the amino acid position within the respective protein. The sequence for the CpRPA3 locus is available from the public data bases (GenBankTM accession number AY994152 [GenBank] ). Inset, CpRPA3 transcript is expressed in sporozoites and contains an intron. Lanes 1 and 5 contain molecular weight markers (M, sizes are indicated at the right of the image). Lane 2 contains the PCR product (405 bp) derived from sporozoite genomic DNA (g). Lane 3 contains a smaller (345-bp) PCR product derived from sporozoite cDNA (c). Lane 4 is a no-template negative control (–). The intron position (relative to the translation) is indicated by an asterisk above the CpRPA3 sequence.

 



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  		FIG. 4.
Binding reactions indicating that CpRPA3 interacts with other CpRPA proteins. Labels are the same as indicated in the legend to Fig. 2. Significant quenching of donor-labeled protein is only observed in the presence of acceptor-labeled MBP-CpRPA3 protein. However, at high concentrations of acceptor-labeled MBP-CpRPA3, the sigmoidal dose-response deviates and becomes linear (reminiscent of nonspecific binding). The acceptor-labeled protein has the expected linear increase in fluorescence in the presence or absence of donor. %Fo = percentage of initial fluorescence. %Fmax = percentage of maximum fluorescence.

 



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  		FIG. 5.
Pull-down assay indicating interaction of CpRPA2 or CpRPA3 with CpRPA1A and CpRPA1B. The MBP or MBP fusions of CpRPA1A (1A) or CpRPA1B (1B) were co-expressed with S-tagged CpRPA2 (S-CpRPA2) and T7-tagged CpRPA3 (T7-CpRPA3) in E. coli. Samples of whole cell lysates (Input) and proteins isolated using amylose resin to pull down MBP proteins from those lysates (Pulldown) were analyzed. Equal amounts of protein were loaded for the pull-down samples, and equal amounts of protein were loaded for the input samples. The S tag was used as a basis for detection of the S-tagged CpRPA2 fusion protein via Western blot (top), and the T7 tag was used as a basis for detection of the T7-CpRPA3 fusion protein. Relative densities of protein bands are shown on the right of blots.

 
Subsequent phylogenetic reconstructions based on 37 sequences and 192 amino acid positions robustly separated all sequences into four major clusters (apicomplexans, plants, animals, and fungi). ML trees reconstructed using PROML and BI methods shared the same topology, in which all apicomplexan RPA1 proteins formed a monophyletic clade (Fig. 7). This observation suggests that all apicomplexan RPA1 homologues (including short and long types) were likely to have originated from a common ancestor. The monophyletic relationship of apicomplexan RPA1 proteins was fully supported by BI-based postrun analysis (posterior probability = 100%). However, within the apicomplexan cluster, RPA1 proteins were apparently grouped based on their types, rather than species, suggesting that the separation of two types of RPA1 proteins might predate the evolutionary expansion of apicomplexan species. The only exception might be the intermediate type of T. gondii RPA1 homologue, which was grouped at the base of a long type cluster, suggesting that this protein might have evolved from the T. gondii long type RPA1 after the separation of T. gondii from other apicomplexans. Another intriguing finding is the plant relationship of apicomplexan RPA1 proteins, although the supporting statistical value is moderate (i.e. posterior probability = 66%).



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  		FIG. 6.
Assessment of the long and short forms of apicomplexan RPA1 proteins. Top, schematic representation of the apicomplexan RPA1 proteins depicting various conserved blocks or functional domains. Common regions include the DNA binding domains and the zinc finger. The long forms contain an extended N terminus. The RFA1-like domains identified by the CD data base and the tRNA-anticodon-like motifs that distinguish the long and short forms are indicated. Bottom, alignment of the apicomplexan RPA1 proteins with the yeast and human RPA1 proteins. The region presented corresponds to the RAD51/RAD52 interaction domain within the tRNA anticodon-like OB fold of the human RPA1 ssDNA binding domain A. The shaded amino acids indicate 50% or greater conservation. Note that all of the short forms possess a deletion within this region.

 
Two Types of RPA1 Genes Are Differentially Expressed at Various Apicomplexan Developmental Stages—The apparent structural difference between the short and long types of apicomplexan RPA1 subunits implies that these two types of RPA1 proteins might play different roles in regulating apicomplexan DNA metabolism. Therefore, we examined the expression patterns of RPA1 transcripts in three apicomplexans. Real time quantitative RT-PCR clearly shows that different RPA1 genes are differentially expressed in C. parvum and T. gondii at various developmental stages (Fig. 8, A and B). This notion is also supported by microarray data of P. falciparum RPA1 genes (Fig. 8C) from a published data base (28, 37). In all three cases, transcripts from one RPA locus were substantially higher than the other(s) during the life cycle of these apicomplexans. For example, in C. parvum, although CpRPA1A displayed a slightly differential expression pattern during its complex life cycle, its expression was always predominant in comparison with that of CpRPA1B. On the other hand, the CpRPA1B transcript periodically increased during intracellular development, suggesting a recurrent need to up-regulate this locus. These data suggest that the various apicomplexan RPA1 subunits may play different roles during parasite development, although further investigation is needed to test this hypothesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we have identified and characterized two distinct types of RPA1 subunits from C. parvum and found that, like human and fungal RPA1 subunits, both CpRPA1A and CpRPA1B maintain a central role as ssDNA-binding proteins. Although their ssDNA binding properties are characteristic of other RPA1 proteins, detailed binding affinity and cooperativity differ from those of the single human RPA1. Among human, plant, and fungal RPA1 proteins, the N-terminal extensions are less conserved and are responsible for interacting with several important regulatory elements (5, 6). However, the short type CpRPA1A (~50 kDa) lacks such an N-terminal extension, suggesting that the function of this protein may be regulated by a mechanism different than the human host.



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  		FIG. 7.
Maximum likelihood (ML) trees inferred from 37 RPA1 protein sequences (192 amino acid positions, –ln L = 6850.7). Statistical supporting values are posterior probabilities (PP) based on a Bayesian inference method (solid circles represent 100% PP values).

 



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  		FIG. 8.
Expression analysis of RPA1 genes from apicomplexans. RPA1 expression levels were based on real time quantitative RT-PCR for C. parvum (relative to those of 18 S rRNA) (A), T. gondii (relative to those of glyceraldehyde-3-phosphate dehydrogenase transcripts) (B), and publicly available data from microarray analysis for P. falciparium (raw data available on the World Wide Web at plasmodb.org) (C). For C. parvum, oocysts (Ooc), sporozoites (Spz), and intracellular stages from an in vitro model at 6, 12, 24, 36, 48, 60, and 72 h postinfection (labeled as 6h to 72h) were examined. For T. gondii, sporozoites, bradyzoites (Bz), and tachyzoites (Tz) were examined. For P. falciparum, data represent sorbitol (S) or temperature (T)-synchronized cultures that contained sporozoites, early ring (ER), late ring (LR), early trophozoites (ETz), late trophozoites (LTz), early schizonts (ESc), late schizonts (LSc), merozoites (Mz), and gametocytes (Gmt). Differential expression is observed for the different RPA1 loci at various developmental stages, and within each species, one transcript predominates at each stage.

 
Although the ORF of CpRPA1B predicts a long type protein (~70 kDa), two types of proteins (~70 and ~45 kDa) may be derived from its transcript. It remains to be determined whether both forms are functional units or if the 70-kDa protein needs to be post-translationally processed to become functional. Nonetheless, functional analysis using recombinant proteins revealed that both the 70- and 45-kDa proteins maintained similar ssDNA binding kinetics, suggesting that the N-terminal extension in CpRPA1B (if present) might play other roles (e.g. functional regulations) rather than ssDNA binding. Similarly, the long type P. falciparum RPA1 locus appears to also encode two proteins. In an earlier report, the short form (~55 kDa) derived from locus PFD0475c, but lacking an N-terminal extension, was characterized from P. falciparum (14). On the other hand, a peptide fragment within the N-terminal extension was detected by an MS-based proteomic analysis (38).

The presence of multiple types of RPA1 subunits appears to be a common feature among apicomplexans. Despite the striking difference between short and long types of apicomplexan RPA1 subunits, all of them appear to have originated from a common ancestor, and one type probably evolved from the other by a gene duplication event before the emergence of the various apicomplexan species. However, it is still unclear whether the gene duplication occurred before or after the separation of apicomplexans from the Alveolata, which also contains dinoflagellates and ciliates. This may only be resolved when RPA1 sequences from dinoflagellates and ciliates become available.

We have also identified RPA2 and RPA3 subunits from C. parvum, P. falciparum, and T. gondii. Using C. parvum as a model, we confirmed that these smaller subunits could specifically interact with each other and with two different types of RPA1 subunits. These data indicate that two distinct types of RPA1 subunits in apicomplexans, or at least in C. parvum, may form heterotrimeric complexes with the same set of RPA2 and RPA3 subunits. The data also suggest that the RPA heterotrimeric complex is probably a universally conserved structure among lower and higher eukaryotes.

Currently, the short type RPA1 subunits have only been found in protozoa, including apicomplexans and a trypanosomatid C. fasciculate (12–16). Why protozoa express a short type RPA1 and why apicomplexans possess two types of RPA1 subunits are intriguing questions. We speculate that the two types of apicomplexan RPA1 proteins may play different roles in DNA metabolism during the complex parasite life and cell cycles. Our gene expression analysis appears to support this idea, since different RPA1 genes in each of the three apicomplexans are differentially expressed. However, further functional studies on the DNA-RPA1 interactions during the parasite life and cell cycles are needed to confirm this speculation.

Apicomplexans are a group of important parasites. In the case of cryptosporidiosis, completely effective treatments are lacking, and Cryptosporidium infection has been responsible for significant morbidity and mortality among AIDS patients (1, 2). Among other apicomplexans (including Plasmodium, Toxoplasma, and Eimeria), effective treatments have been available. However, drug-resistant strains are emerging rapidly in the field. Thus, there is an urgent need for the development of new drugs. Because DNA metabolism is essential to all organisms, factors involved in DNA and chromatin metabolism have been utilized or proposed as potentially useful targets for chemotherapeutic exploration (15–17, 34, 39, 40). Because the structure and function of RPA proteins differ significantly between apicomplexans and their hosts, we propose that RPA may be explored as a novel target for the development of new anti-apicomplexan drugs.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant R21 AI055278 (to G. Z.). This work was also supported in part by American Heart Association Grant 0235424Z (to W. J. S.).

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY994152 [GenBank] . Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental information on T. gondii RPA predicted proteins. Back

** To whom correspondence should be addressed: Dept. of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, 4467 TAMU, College Station, TX 77843. Tel.: 979-865-6981; Fax: 979-845-9972; E-mail: gzhu{at}cvm.tamu.edu.

1 The abbreviations used are: RPA, replication protein A; BI, Bayesian inference; FRET, fluorescence resonance energy transfer; MBP, maltose-binding protein; ML, maximum likelihood; OB fold, oligonucleotide/oligosaccharide binding fold; ssDNA, single-stranded DNA; ORF, open reading frame; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank David New for technical help in constructing the CpRPA1B N-terminal deletion mutant. The CpRPA3 gene sequence was originally identified from the C. parvum genome sequence project at the University of Minnesota supported by the National Institutes of Health (U01 AI046397 [GenBank] ). Preliminary gene sequences of P. falciparum and T. gondii were obtained from their genome data bases accessible on the World Wide Web at plasmodb.org and toxodb. org, respectively. The plasmodb data base also contains gene expression data utilized in this study.



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 ABSTRACT
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