1 Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA
2 Faculty of Genetics Program, Texas A&M University, 4467 TAMU, College Station, TX 77843, USA
Correspondence
Guan Zhu
Gzhu{at}cvm.tamu.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this article is AY219916.
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INTRODUCTION |
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The biological and biochemical features of RPA proteins have been more intensively investigated in yeast and humans than any other organisms including protists, the most divergent taxonomic group of unicellular species. Although there are a limited number of reports investigating the function of the short-type RPA1 subunit in protists, virtually nothing is known about the function of RPA2. The only identification of a protist RPA2 is the 28 kDa protein from the cytosol of the trypanosomatid parasite Crithidia fasciculata (Brown et al., 1994). In contrast to RPA1 proteins that are relatively conserved among various species, the RPA2 subunit appears to be extremely poorly conserved among all organisms (see below for details), which poses an extra burden on the identification and functional confirmation of protist RPA2 proteins. How this group of divergent proteins plays the same structural and functional roles in various organisms is an intriguing question.
The apicomplexan protist Cryptosporidium parvum is globally recognized as an intracellular parasite of humans and animals that can cause self-limiting diarrhoea in immunocompetent individuals or life-threatening, prolonged opportunistic infections in immunocompromised patients. To date, there is still no effective treatment available for C. parvum infections (Griffin et al., 1998; Okhuysen & Chappell, 2002
; Tzipori & Griffiths, 1998
). We have previously identified and characterized two distinct, short-type RPA1 subunits from the parasite (humans have only one RPA1), indicating that protists may utilize a different mechanism in regulating DNA metabolism (Millership & Zhu, 2002
; Zhu et al., 1999
). In the present study, we have identified an RPA2 homologue (termed CpRPA2) from the C. parvum genome project. Despite its weak homology to other eukaryotic RPA2 proteins, the ssDNA-binding property and phosphorylation ability of CpRPA2 were verified and characterized.
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METHODS |
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Molecular cloning and heterogeneous expression of CpRPA2.
Using eukaryotic RPA2 protein sequences as a query, an RPA2 homologue was identified from the ongoing C. parvum genome project at the University of Minnesota (MN, USA) and designated CpRPA2. The entire putative open reading frame (ORF) of the CpRPA2 gene was cloned by PCR and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein using the following primers: RPA2-F01 (5'-gcg aat tcA TGA ATT TTG GTG AAA ATA AT-3') and RPA2-R01 (5'-gcg aat tcT TAA TAT CCA GTA GCT CTC CAA GT-3') (lower-case letters represent artificially added EcoRI linkers). The ORF was amplified from C. parvum genomic DNA using a high-fidelity Pfu DNA polymerase (Stratagene) and cloned into a pMAL-p2x vector that contains a signal peptide to lead the MBP fusion protein into the bacterial periplasmic space (New England Biolabs). The sequence was confirmed in both directions by automated sequencing performed at Texas A&M University Gene Technologies Laboratory using an ABI PRISM 377 system (Applied Biosystems). The plasmids containing correctly oriented CpRPA2 sequence were transformed into the Rosetta strain of E. coli (Novagen). Expression and purification of MBP fusion proteins was performed as described by the manufacturer, except that IPTG-induced expression was performed at a low temperature (16 °C) overnight. The MBP fusion protein was observed to interfere with assays and was thus removed by the addition of factor X. Following cleavage, the free MBP-tag was removed by affinity chromatography with amylose resin, thus leaving the purified recombinant CpRPA2 protein (rCpRPA2).
Antibody production and purification.
MBP-fused CpRPA2 was freshly emulsified with TiterMax Gold (TiterMax Gold USA) prior to each immunization. Polyclonal antibodies to MBPCpRPA2 were raised in a specific pathogen-free rabbit that was initially immunized with 200 µg of antigen. Booster immunizations (100 µg each) were performed later at 30 and 60 days, respectively. Rabbit serum was collected prior to and after the immunization protocol. A negative and positive purification procedure was employed for the isolation of rCpRPA2-specific antibodies to ensure specificity using a previously reported protocol with slight modifications (Benet & van Cutsem, 2002). Our protocol was performed in three separate procedures: the first and second steps were used to remove the antiserum specific to the MBP-tag and non-specific binding with the aid of E. coli supernatants, respectively, while the third step was designed to purify the rCpRPA2-specific antibodies. Briefly, the MBP-tag (500 µg total) was immobilized onto a nitrocellulose membrane (5 cmx5 cm) and, following drying, the membrane was blocked with 3 % BSA in TBS containing 10 mM Tris/HCl and 500 mM NaCl for 2 h and washed twice with TBS for 10 min. Rabbit antiserum diluted (1 : 100) in 1 % BSA/TBS was applied to the membrane and gently agitated for 3 h. The second phase of the purification utilized supernatants of E. coli prepared by sonication and applied to a second nitrocellulose membrane (5 cmx5 cm). The membrane was air-dried and was washed three times in TBS and blocked as described above. The antiserum from the first phase of purification was applied to the second nitrocellulose membrane and incubated for 2 h with gentle agitation. The MBPCpRPA2 (200 µg total protein) was then applied to a third nitrocellulose membrane (5 cmx5 cm) as described above. The membrane was incubated in the antiserum preparation from the second step for 3 h and washed five times in TBS for 10 min with gentle agitation. The rCpRPA2-specific antibodies were eluted by incubating the membrane in 10 ml of 0·2 M glycine/HCl (pH 2·5) for 3 min. The eluted antibody solution was neutralized by the addition of 1·7 ml of 1 M Tris/HCl (pH 8·8) containing 7 % BSA and used at 1/100 to 1/1000 final dilutions. All steps were performed at room temperature. Pre-serum was prepared in the same manner and failed to elicit a response both in Western blots and by indirect immunofluorescence.
Western blot analysis.
This was performed using the affinity-purified, rCpRPA2-specific rabbit polyclonal antibody that was secondarily labelled with a monoclonal antibody against rabbit IgG conjugated with horseradish peroxidase (HRP) (Sigma Chemical). Approximately 1x107 oocysts per well were lysed in loading buffer containing a protease inhibitor cocktail (Sigma Chemical) at 95 °C for 5 min. Insoluble material was removed by centrifugation and soluble proteins were resolved on 10 % SDS-PAGE gels and transferred to nitrocellulose as described by mini trans-blot electrophoretic transfer (Bio-Rad). All immunological processing was performed as described by the Bio-Rad Western blot protocol. Briefly, following transfer of proteins, blots were incubated in 3 % gelatin in TBS (20 mM Tris/HCl, pH 7·5; 500 mM NaCl) for 1 h. The primary antibody (either purified immune serum or pre-serum) was diluted with 1 % gelatin in Tris/Tween 20-buffered saline (TTBS: 20 mM Tris/HCl, pH 7·5; 500 mM NaCl; 0·05 % Tween 20) and incubated for 1 h at 37 °C. Following washing with Tris/citrate-buffered saline (TCBS: 20 mM citrate, pH 5·5; 500 mM NaCl; 0·05 % Tween 20), the blots were incubated in a secondary antibody conjugated to HRP (diluted in TCBS) for 1 h. Prior to analysis, blots were washed with TTBS and TBS, respectively, and colour development was performed with 3,3'-diaminobenzidine (Sigma Chemical) as substrate.
Semi-quantitative RT-PCR.
This was performed similar to a previously described protocol (Abrahamsen & Schroeder, 1999). However, the quantity of products was densitometrically (rather than radioactively) measured in agarose gels. Total RNA was isolated from C. parvum free sporozoites and intracellular stages developed in HCT-8 cells in vitro for various times (372 h) using RNeasy kit (Qiagen). All RNA samples were subject to intensive RNase-free DNase digestion until no products could be amplified by PCR. Since RNA samples isolated from intracellular parasites were mixed with host and parasite RNA, all samples were first normalized using C. parvum 18S rRNA by a semi-quantitative RT-PCR with a pair of previously described primers (i.e. 995F and 1206R) (Abrahamsen & Schroeder, 1999
). Twenty thermal cycles were employed so that the densities of RT-PCR amplicons could be measured within linear ranges in agarose gels. The RNA concentrations from various samples were adjusted to produce comparable amounts of C. parvum 18S rRNA amplicons by RT-PCR. The adjusted amounts of total RNA were then used for RT-PCR amplification of the CpRPA2 transcript by 23 thermal cycles using appropriate primer sets. The density of each amplicon was determined using GENETOOLS software v. 3.00.22 (Hitachi Software Engineering) and its relative level of transcripts at each time point was expressed as the ratio of signals between CpRPA2 and 18S rRNA amplicons.
ssDNA-binding assay.
The standard ssDNA binding reactions were performed in 15 µl Tris/HCl buffer (pH 7·4, 20 mM) containing 150 mM NaCl, 25 % glycerol and 1 mM ZnCl2, which contained the indicated concentration of rCpRPA2 (Eckerich et al., 2001; Millership & Zhu, 2002
). The 5'-biotinylated oligonucleotides [(dT)5, (dT)10, (dT)15, (dT)17, (dT)20, (dT)22, (dT)25, (dT)30 and (dT)40 (5 fmol each unless stated)] were combined with the protein and the reaction was incubated at 25 °C for 30 min. Control groups included MBP-tag (a non-specific protein) plus oligonucleotide and the oligonucleotides alone (no protein). The retardation of the oligonucleotideprotein complex was resolved on 6 % native polyacrylamide gels, which were buffered with 50 mM Tris/borate (pH 7·5). Following electrophoresis, reactions were transferred onto Zeta-Probe GT nylon membranes (Bio-Rad) and the free and protein-bound oligonucleotides were detected using a Pierce LightShift chemiluminescent electrophoretic mobility shifting assay (EMSA) kit (Pierce).
rCpRPA2 phosphorylation in vitro by DNA-dependent protein kinase (DNA-PK).
The phosphorylation of rCpRPA2 was investigated according to a previously described protocol with slight modifications (Brush & Kelly, 2000; Brush et al., 2001
). Briefly, a reaction mixture (50 µl) in a buffer (50 mM HEPES; 100 mM KCl; 10 mM MgCl2; 0·2 mM EGTA; 1 mM DTT) containing BSA (2·5 µg), calf thymus DNA (0·01 µg), [
-32P]ATP (100 µCi; 3·70 MBq), rCpRPA2 (0·02 µg) and DNA-PK (25 U) was first gently agitated at 30 °C for 30 min and halted by the addition of SDS-PAGE loading buffer. One of the DNA-PK-treated samples was heat-inactivated without adding loading buffer, followed by the addition of calf intestinal alkaline phosphatase (10 U) to test whether the newly phosphorylated rCpRPA2 could be dephosphorylated. The samples were heated at 95 °C for 5 min and subjected to SDS-PAGE (8 %), and the separated proteins transferred to nitrocellulose membranes. The resulting blot was analysed by autoradiography. Reactions without dsDNA and/or DNA-PK were included as controls.
Immunofluorescence microscopy.
C. parvum sporozoites were prepared by excystation of fresh oocysts in PBS containing 0·5 % trypsin and 0·25 % taurodeoxycholic acid for 1 h at 37 °C. Excysted free sporozoites were washed three times in PBS by centrifugation, fixed in a PBS-buffered formalin solution for at least 15 min and washed again in PBS and water (twice each). Sporozoites suspended in water were then adhered to microscope slides pre-coated with 0·1 % poly-L-lysine, air-dried and extracted with cold methanol/acetone (1 : 1, v/v) for 5 min at 20 °C. Following blocking in PBS containing 5 % BSA and 0·05 % Tween 20 for 1 h at room temperature, samples were incubated in primary antibodies diluted in PBS containing 1 % BSA and 0·05 % Tween 20 for 1 h. Each sample was then incubated with a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody (monoclonal anti-rabbit-TRITC; Sigma Chemical). Wash steps between each stage were accomplished with PBS containing 0·05 % Tween 20. Slides were mounted using anti-quenching medium (Molecular Probes) and viewed with a BX51 Olympus microscope equipped with a FITC/TRITC filter set and photographed with an Olympus PM10SP camera.
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RESULTS |
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The MBP-tag was found to interfere with the ssDNA-binding property of CpRPA2 and thus was removed from the fusion protein. MBP-free rCpRPA2 was purified and utilized to study the ssDNA-binding properties by EMSA. First, we demonstrated that rCpRPA2 was able to bind to biotinylated (dT)40 oligonucleotides (Fig. 4a). We also observed a concentration-dependent binding activity of rCpRPA2 to the oligonucleotide (data not shown). In a second study, the ability of rCpRPA2 to bind to various lengths of ssDNA [(dT)1540] was investigated, and this revealed that rCpRPA2 was able to bind oligo(dT) of
17 nt in length (Fig. 4b
). These observations taken together verified that the CpRPA2-encoded protein belongs to the family of eukaryotic ssDNA-binding proteins.
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DISCUSSION |
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RPA participates in essential roles for DNA metabolism, which include replication, repair and recombination. All animals, plant and fungi studied to date possess only one type of RPA trimeric complex, with subunits of approximately 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3). The DNA binding activity of RPA has been isolated to RPA1 and RPA2 (Iftode et al., 1999; Wold, 1997
). In addition to ssDNA-binding, RPA1 has been shown to interact with a number of cellular proteins that regulate the cell cycle, DNA repair and recombination. Whereas RPA2 is thought to have a regulatory function, thought to be controlled by phosphorylation (Brill & Bastin-Shanower, 1998
), the role of RPA3 still remains ambiguous, but studies indicate that it is probably associated with assembly and stabilization of the trimeric subunit (Iftode et al., 1999
; Wold, 1997
).
While C. parvum is a eukaryote, our studies have indicated that its DNA-binding proteins differ from other known eukaryotic RPA complexes. Indeed, in contrast to its human and animal hosts, C. parvum possesses two distinct RPA1 subunits (CpRPA1 and CpRPA1B) (Millership & Zhu, 2002; Zhu et al., 1999
). These two subunits are also significantly smaller than other eukaryotic RPA1 subunits and possess different DNA binding properties in vitro. Our preliminary localization analyses indicate that CpRPA1 and CpRPA1B probably act independently through the parasite's life cycle (data not shown). The identification of CpRPA2 provides us with the opportunity to further understand the structure and action of the RPA complex during the life cycle of C. parvum. However, it is unclear whether CpRPA2 interacts with one or both of CpRPA1 or CpRPA1B subunits. Indeed, we have been unsuccessful in identifying a second possible RPA2-encoding gene from the complete C. parvum genome. The search of RPA2 homologues from the complete P. falciparum genome was not successful, which was probably due to the high divergence of this protein or possible intron interruption(s) present in the P. falciparum homologue(s). However, one can not rule out the possibility that, although P. falciparum possesses a RPA1 homologue (i.e. PfRPA1), it probably lacks a second ssDNA-binding protein, such as RPA2. This notion is in part supported by a previous study which showed only a single species of protein (PfRPA1) from crude nuclear extract was able to form an ssDNAprotein complex (Voss et al., 2002
).
Unlike RPA1 which has defined regions of homology, no apparent regions have been identified to date within RPA2, except for two short motifs at the N and C termini (Fig. 1). However, in general, RPA2 has three domains: the N terminus contains the phosphorylation site, the central domain is the DNA-binding region and the C-terminal is the binding domain for other proteins (Iftode et al., 1999
). Comparison of the characterized RPA2 proteins and annotated homologues from the genomes of various organisms supports the notion that RPA2 is a group of highly divergent molecules (Fig. 1
). The lack of an in vitro culture system for C. parvum complicates our ability to investigate the replication pathways of this unique protist and thus the use of recombinant proteins is the only feasible way to perform functional protein studies on this parasite. In this study, we have confirmed that rCpRPA2 is an active ssDNA-binding protein and that it can be phosphorylated by DNA-PK (Fig. 4a
). In addition, the phosphorylation appears to inhibit binding of rCpRPA2 to ssDNA in vitro (Fig. 4b
). The functional significance of the N-terminal RPA2 phosphorylation is not fully understood (Iftode et al., 1999
). In some cases when RPA2 is phosphorylated it becomes disassociated from RPA1, but this is not always the case (Treuner et al., 1999a
, b
). Indeed, deletion of the human RPA2 (HsRPA) N-terminal region (containing the phosphorylation sites) has no effect on replication in SV40 DNA replication (Iftode et al., 1999
; Wold, 1997
). However, recent data indicated that RPA2 phosphorylation might play an important role in DNA repair (Iftode et al., 1999
; Wold, 1997
). HsRPA2 becomes hyper-phosphorylated when human cells are damaged by UV radiation, and this coincides with the inability to support SV40 DNA replication in vitro (Boubnov & Weaver, 1995
; Carty et al., 1994
; Fried et al., 1996
). Although our data clearly indicated that rCpRPA2 could be phosphorylated in vitro by DNA-PK, it was unclear whether or how the phosphorylation of CpRPA2 might occur in vivo. Nonetheless, the ability of rCpRPA2 to serve as a substrate for phosphorylation clearly pointed out a possible new direction to further investigate the function and regulation of this important parasite protein.
Interest in the HsRPA complex stems from the importance of this trimer in DNA metabolism, and thus cancer biology. To date, several anti-cancer drugs have been identified that interact with the RPA complex (Liu et al., 2000; Peters et al., 2001
). One of these, tirapazamine, is a hypoxia-activated cytotoxic agent which is currently in phase III clinical studies and has been localized to RPA2 (Peters et al., 2001
). Indeed, monoclonal antibodies against HsRPA2 inhibit DNA replication, by impeding RPA stimulation of DNA polymerase alpha, but do not stop the RPA complex from binding to DNA (Basilion et al., 1999
). Alternative methods have also been utilized in the form of anti-sense technologies to inhibit the RPA complex (Basilion et al., 1999
; Gomes & Wold, 1996
; Kenny et al., 1990
). Together with the evidence that C. parvum possesses a novel CpRPA2 and two distinct, short-type large subunits (Millership & Zhu, 2002
), all of which differ significantly from its host, one could speculate that the parasite's DNA replication proteins may serve as a novel target for drugs against cryptosporidiosis and possibly other apicomplexan-based diseases.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Basilion, J. P., Schievella, A. R., Burns, E., Rioux, P., Olson, J. C., Monia, B. P., Lemonidis, K. M., Stanton, V. P., Jr & Housman, D. E. (1999). Selective killing of cancer cells based on loss of heterozygosity and normal variation in the human genome: a new paradigm for anticancer drug therapy. Mol Pharmacol 56, 359369.
Benet, C. & van Cutsem, P. (2002). Negative purification method for the selection of specific antibodies from polyclonal antisera. Biotechniques 33, 1050, 10521054.
Boubnov, N. V. & Weaver, D. T. (1995). scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase. Mol Cell Biol 15, 57005706.[Abstract]
Brill, S. J. & Bastin-Shanower, S. (1998). Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A. Mol Cell Biol 18, 72257234.
Brown, L. M. & Ray, D. S. (1997). Cell cycle regulation of RPA1 transcript levels in the trypanosomatid Crithidia fasciculata. Nucleic Acids Res 25, 32813289.
Brown, G. W., Hines, J. C., Fisher, P. & Ray, D. S. (1994). Isolation of the genes encoding the 51-kilodalton and 28-kilodalton subunits of Crithidia fasciculata replication protein A. Mol Biochem Parasitol 63, 135142.[CrossRef][Medline]
Brush, G. S. & Kelly, T. J. (2000). Phosphorylation of the replication protein A large subunit in the Saccharomyces cerevisiae checkpoint response. Nucleic Acids Res 28, 37253732.
Brush, G. S., Kelly, T. J. & Stillman, B. (1995). Identification of eukaryotic DNA replication proteins using simian virus 40 in vitro replication system. Methods Enzymol 262, 522548.[Medline]
Brush, G. S., Clifford, D. M., Marinco, S. M. & Bartrand, A. J. (2001). Replication protein A is sequentially phosphorylated during meiosis. Nucleic Acids Res 29, 48084817.
Carty, M. P., Zernik-Kobak, M., McGrath, S. & Dixon, K. (1994). UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein. EMBO J 13, 21142123.[Abstract]
Eckerich, C., Fackelmayer, F. O. & Knippers, R. (2001). Zinc affects the conformation of nucleoprotein filaments formed by replication protein A (RPA) and long natural DNA molecules. Biochim Biophys Acta 1538, 6775.[Medline]
Fairman, M. P. & Stillman, B. (1988). Cellular factors required for multiple stages of SV40 DNA replication in vitro. EMBO J 7, 12111218.[Abstract]
Fried, L. M., Koumenis, C., Peterson, S. R. & 8 other authors (1996). The DNA damage response in DNA-dependent protein kinase-deficient SCID mouse cells: replication protein A hyperphosphorylation and p53 induction. Proc Natl Acad Sci U S A 93, 1382513830.
Gomes, X. V. & Wold, M. S. (1996). Functional domains of the 70-kilodalton subunit of human replication protein A. Biochemistry 35, 1055810568.[CrossRef][Medline]
Griffin, R. J., Dunwoody, S. & Zabala, F. (1998). Public reliance on risk communication channels in the wake of a Cryptosporidium outbreak. Risk Anal 18, 367375.[Medline]
Henricksen, L. A., Carter, T., Dutta, A. & Wold, M. S. (1996). Phosphorylation of human replication protein A by the DNA-dependent protein kinase is involved in the modulation of DNA replication. Nucleic Acids Res 24, 31073112.
Iftode, C., Daniely, Y. & Borowiec, J. A. (1999). Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol 34, 141180.
Kenny, M. K., Schlegel, U., Furneaux, H. & Hurwitz, J. (1990). The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J Biol Chem 265, 76937700.
Kim, C., Snyder, R. O. & Wold, M. S. (1992). Binding properties of replication protein A from human and yeast cells. Mol Cell Biol 12, 30503059.[Abstract]
Liu, J. S., Kuo, S. R., McHugh, M. M., Beerman, T. A. & Melendy, T. (2000). Adozelesin triggers DNA damage response pathways and arrests SV40 DNA replication through replication protein A inactivation. J Biol Chem 275, 13911397.
Millership, J. J. & Zhu, G. (2002). Heterogeneous expression and functional analysis of two distinct replication protein A large subunits from Cryptosporidium parvum. Int J Parasitol 32, 14771485.[CrossRef][Medline]
Okhuysen, P. C. & Chappell, C. L. (2002). Cryptosporidium virulence determinants are we there yet? Int J Parasitol 32, 517525.[CrossRef][Medline]
Peters, K. B., Wang, H., Brown, J. M. & Iliakis, G. (2001). Inhibition of DNA replication by tirapazamine. Cancer Res 61, 54255431.
Treuner, K., Findeisen, M., Strausfeld, U. & Knippers, R. (1999a). Phosphorylation of replication protein A middle subunit (RPA32) leads to a disassembly of the RPA heterotrimer. J Biol Chem 274, 1555615561.
Treuner, K., Okuyama, A., Knippers, R. & Fackelmayer, F. O. (1999b). Hyperphosphorylation of replication protein A middle subunit (RPA32) in apoptosis. Nucleic Acids Res 27, 14991504.
Tzipori, S. & Griffiths, J. K. (1998). Natural history and biology of Cryptosporidium parvum. Adv Parasitol 40, 536.[Medline]
Voss, T. S., Mini, T., Jenoe, P. & Beck, H.-P. (2002). Plasmodium falciparum possesses a cell cycle-regulated short type replication protein A large subunit encoded by an unusual transcript. J Biol Chem 277, 1749317501.
Wold, M. S. (1997). Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66, 6192.[CrossRef][Medline]
Wold, M. S. & Kelly, T. (1988). Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A 85, 25232527.[Abstract]
Wold, M. S., Weinberg, D. H., Virshup, D. M., Li, J. J. & Kelly, T. J. (1989). Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem 264, 28012809.
Zhu, G., Marchewka, M. J. & Keithly, J. S. (1999). Cryptosporidium parvum possesses a short-type replication protein A large subunit that differs from its host. FEMS Microbiol Lett 176, 367372.[CrossRef][Medline]
Zhu, G., Marchewka, M. J., Woods, K. M., Upton, S. J. & Keithly, J. S. (2000). Molecular analysis of a Type I fatty acid synthase in Cryptosporidium parvum. Mol Biochem Parasitol 105, 253260.[CrossRef][Medline]
Received 16 October 2003;
revised 27 January 2004;
accepted 4 February 2004.