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Conservation of Structure and Function of DNA Replication Protein a in the Trypanosomatid Crithidia Fasciculata GRANT W

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Conservation of Structure and Function of DNA Replication Protein a in the Trypanosomatid Crithidia Fasciculata GRANT W Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 10227-10231, November 1992 Biochemistry Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata GRANT W. BROWN*, THOMAS E. MELENDYt, AND DAN S. RAY*4 *Molecular Biology Institute and tDepartment of Biology, University of California, Los Angeles, CA 90024; and tCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 Communicated by R. L. Sinsheimer, July 6, 1992 ABSTRACT Human replication protein A (RP-A) is a replication proteins from higher eukaryotes, whether they are three-subunit protein that is required for simian virus 40 regulated in a cell cycle-dependent fashion, and whether their (SV40) replication in vitro. The trypanosome homologue of properties can be exploited as targets for therapeutic drugs. RP-A has been purified from Crithidiafasciculat. It is a 1:1:1 Neither proteins involved in initiation ofDNA replication nor complex of three polypeptides of 51, 28, and 14 kDa, binds proteins involved in the cell cycle regulation of replication single-stranded DNA via the large subunit, and is localized have been identified. within the nucleus. C. fasciculdta RP-A substitutes for human Replication protein A [RP-A; also called RF-A or human RP-A in the large tumor antigen-dependent unwinding of the single-stranded DNA-binding protein (SSB)] was first isolated SV40 origin of replication and stimulates both DNA synthesis from a protein fraction ofhuman cells required for the in vitro and DNA priming by human DNA polymerase a/primase, but replication of simian virus 40 (SV40) (9-11). It is a three- it does not support efficient SV40 DNA replication in vitro. This subunit, multifunctional SSB involved in both the initiation extraordinary conservation of structure and function between and elongation phases of SV40 replication. With topoisomer- human and trypanosome RP-A suggests that the mechanism of ase I and the SV40 large tumor antigen (T-Ag), RP-A partic- DNA replication, at both the initiation and the elongation level, ipates in the unwinding ofthe SV40 origin ofreplication during is conserved in organisms that diverged from the main eukary- the initiation of DNA replication (12-14). RP-A stimulates otic lineage very early in evolution. DNA synthesis by DNA polymerases a and a (15-17), sug- gesting that RP-A functions in the elongation phase of DNA Trypanosomes are protozoan parasites of considerable med- replication in vitro. RP-A is present in the yeast Saccharomy- ical and economic importance; they cause serious disease in ces cerevisiae (18), indicating that its activity is evolutionarily humans and livestock. They are characterized by the presence conserved. In both yeast and human cells, RP-A is posttrans- ofa kinetoplast, a massive network ofcatenated circular DNA lationally modified by phosphorylation of the middle subunit molecules located at the base ofthe flagellum within the single in a cell cycle-dependent manner (19), suggesting a role for mitochondrion of these cells. Trypanosomes diverged from RP-A in the cell cycle regulation ofDNA replication. Extracts the major eukaryotic lineage very early in the evolutionary from cells in G1 phase, the phase in which RP-A is unphos- history of eukaryotic cells relative to the separation of the phorylated, have lower activity in unwinding the SV40 origin "higher" evolutionary groups, the plants, animals, and fungi of replication and are 10 times less efficient at initiating SV40 (1, 2). Studies oftranscription in trypanosomes have revealed replication, when compared to extracts from cells in S or G2 a number of unusual features, including trans-splicing, a phase, the phases during which RP-A is phosphorylated (20). process by which discontinuously synthesized precursor A link between a regulatory role for RP-A and the cell cycle RNAs are spliced to yield mature messenger RNAs, and RNA control machinery is suggested by the observation that G1 editing, which involves the insertion and deletion of uridine extracts can be activated by a fraction that contains a kinase residues in mitochondrial RNA transcripts. Studies of DNA homologous to the p34cdc2 kinase (21). The genes encoding the replication in trypanosomes have focused mainly on the ki- three subunits of RP-A in yeast are all essential for viability netoplast minicircle DNA, a molecule unique among mito- (22), indicating the essential nature of the functions of RP-A. chondrial DNAs for its high copy number and its cell cycle- In this report, the identification and characterization of the regulated replication (for review, see refs. 3 and 4). The trypanosome homologue of RP-A are described. biochemical mechanism and the regulation of nuclear DNA replication in trypanosomes are poorly understood. Only one protein expected to be involved in nuclear DNA replication MATERIALS AND METHODS has been purified to homogeneity from a trypanosome, a type CeU Growth. C. fasciculata Cf-Cl was grown, harvested, I topoisomerase from Crithidiafasciculata (5). Two RNase H and stored frozen at -750C as described (23). activities in crude extracts of C. fasciculata have been de- Purification of RP-A. RP-A was purified from C. fascicu- scribed (6) and seem to be similar to the class I and class II lata as described for the purification ofRP-A from yeast (18), RNase H enzymes of higher eukaryotes. Two DNA polymer- with several modifications. Fifty grams offrozen cells (-5 X ases have been partially purified from C. fasciculata and 1011 cells) was thawed in 250 ml of50 mM Hepes (pH 7.5), 10 Trypanosoma brucei (7, 8). These proteins are similar to DNA mM EDTA, 0.5 M NaCl, 2.5 mM 2-mercaptoethanol, leu- polymerases a and ( of higher eukaryotes, but they were peptin (2 ug/ml), pepstatin A (1 ug/ml), 1 mM phenylmeth- found to differ from their mammalian homologues in their ylsulfonyl fluoride, and 10 mM benzamidine. After homog- response to several DNA polymerase inhibitors (7). Little else enization with a motor-driven Teflon pestle, the lysate was is known of other proteins involved in chromosomal DNA stirred for 30 min at 0C. The lysate was clarified by centrif- replication in trypanosomes and their biochemical proper- ugation in a Beckman Ti45 rotor (40,000 rpm for 30 min at ties-for example, whether they are homologous to known 20C). The supernatant was filtered through Miracloth and The publication costs of this article were defrayed in part by page charge Abbreviations: RP-A, replication protein A; SV40, simian virus 40; payment. This article must therefore be hereby marked "advertisement" ssDNA, single-stranded DNA; SSB, single-stranded DNA-binding in accordance with 18 U.S.C. §1734 solely to indicate this fact. protein; T-Ag, large tumor antigen. 10227 Downloaded by guest on September 23, 2021 10228 Biochemistry: Brown et al. Proc. Natl. Acad. Sci. USA 89 (1992) centrifuged for an additional 90 min at 40,000 rpm at 20C in nitroblue tetrazolium and 5-bromo-4-chloro-3-indoylyl phos- a Ti45 rotor. The clear amber supernatant was loaded onto a phate (Promega). 2.5 x 3 cm single-stranded DNA (ssDNA)-celiulose (Sigma) Immunocytochemistry. Immunolocalizations were per- column equilibrated in 25 mM Hepes (pH 7.5), 1 mM EDTA, formed essentially as described (5), using a 200-fold dilution 10% (vol/vol) glycerol, 0.02% (wt/vol) Brij 58, 2.5 mM of antiserum raised against C. fasciculata RP-A. 2-mercaptoethanol, leupeptin (1 ,ug/ml), pepstatin A (0.5 ,ug/ml), and 0.25 mM phenylmethylsulfonyl fluoride (buffer A) containing 0.5 M NaCl. The column was washed with 3 vol RESULTS of buffer A containing 0.75 M NaCl, and the protein was Purification of RP-A From the Trypanosomatid C. fascicu- eluted with buffer A containing 1.5 M NaCl and 50%o (vol/vol) iota. The C. fasciculata homologue of RP-A was purified by ethylene glycol. Protein-containing fractions were pooled using ssDNA-cellulose and Mono Q column chromatogra- and dialyzed against 25 mM imidazole hydrochloride (pH phy. As is the case with RP-A from S. cerevisiae and human 7.5), 0.1 mM EDTA, 10%6 glycerol, 0.02% Brij 58, 2.5 mM cells, the protein from C.fasciculata bound tightly to ssDNA, 2-mercaptoethanol, leupeptin (1 ,g/ml), pepstatin A (0.5 requiring greater than 0.75 M NaCl for elution. The Mono Q pg/ml), and 0.25 mM phenylmethylsulfonyl fluoride (buffer column removed trace contaminants present after the ssDNA B) containing 10 mM NaCl. The dialysate was clarified by column and concentrated the RP-A. Fig. la shows SDS/ passage through a 0.2-,um nitrocellulose filter and applied to PAGE analysis of each step in the purification and Mono Q a Mono Q column (HR 5/5; Pharmacia). The column was column fractions 1-10. The peak of C. fasciculata RP-A washed with S ml ofbuffer B containing 10mM NaCl, and the eluted from the Mono Q column at 125 mM NaCI, in protein was eluted with a 20-ml linear gradient from 10 mM fractions 5 and 6. These fractions contained three polypep- to 400 mM NaCl in buffer B. This procedure yielded 300 I&g tides with molecular masses of 51 28 and 14 kDa of RP-A, as determined by a modified Bradford assay (24), kDa, kDa, using bovine serum albumin as a standard. The preparation was 94% homogeneous as judged by scanning densitometry V. of Coomassie brilliant blue-stained polyacrylamide gels. The ...F molar ratio of the subunits was also determined by densi- tometry. Reaction Conditions. SV40 origin unwinding assays (25 ,ul) were performed as described (13), with incubation at 37°C for 30 min. Reactions were terminated, and reaction mixtures were processed for electrophoresis as described (15) and electrophoresed for 12 hr at 25 V through a 1.5% agarose gel in 40 mM Tris acetate, pH 7.5/1 mM EDTA.
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