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• W1965602008 abstract "Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation. Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is a remarkable DNA structure that contains, in the species Crithidia fasciculata, 5000 topologically linked duplex DNA minicircles. Their replication initiates at two conserved sequences, a dodecamer, known as the universal minicircle sequence (UMS), and a hexamer, which are located at the replication origins of the minicircle L and H strands, respectively. A UMS-binding protein (UMSBP) binds specifically the 12-mer UMS sequence and a 14-mer sequence that contains the conserved hexamer in their single-stranded DNA conformation. In vivo cross-linking analyses reveal the binding of UMSBP to kinetoplast DNA networks in the cell. Furthermore, UMSBP binds in vitro to native minicircle origin fragments, carrying the UMSBP recognition sequences. UMSBP binding at the replication origin induces conformational changes in the bound DNA through its folding, aggregation and condensation. Kinetoplast DNA (kDNA) 4The abbreviations used are: kDNA, kinetoplast DNA; UMS, universal minicircle sequence; UMSBP, UMS binding protein; L-strand, the light DNA strand of kinetoplast DNA minicircle; H-strand, the heavy DNA strand of kinetoplast DNA minicircles; oriL, replication origin of the minicircle light strand; oriH, replication origin of the minicircle heavy strand; OriA and OriB, the two origin regions in the C. fasciculata kDNA minicircle, each containing an oriL and an oriH site; WT, wild type; EMSA, electrophoretic mobility shift analysis; SPR, surface plasmon resonance; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 4The abbreviations used are: kDNA, kinetoplast DNA; UMS, universal minicircle sequence; UMSBP, UMS binding protein; L-strand, the light DNA strand of kinetoplast DNA minicircle; H-strand, the heavy DNA strand of kinetoplast DNA minicircles; oriL, replication origin of the minicircle light strand; oriH, replication origin of the minicircle heavy strand; OriA and OriB, the two origin regions in the C. fasciculata kDNA minicircle, each containing an oriL and an oriH site; WT, wild type; EMSA, electrophoretic mobility shift analysis; SPR, surface plasmon resonance; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is a unique extrachromosomal DNA found in the single mitochondrion of trypanosomatids. In the species Crithidia fasciculata the kDNA network consists of ∼5000 duplex DNA minicircles of 2.5 kbp and 50 maxicircles of 37 kbp that are interlocked topologically to form a DNA network (1Ray D.S. Plasmid. 1987; 17: 177-190Crossref PubMed Scopus (45) Google Scholar, 5Shlomai J. Curr. Mol. Med. 2004; 4: 623-647Crossref PubMed Scopus (117) Google Scholar). Maxicircles contain mitochondrial genes, encoding mitochondrial proteins and rRNA. Minicircles encode for guide RNAs that function in the process of mitochondrial mRNA editing (6Stuart K. Allen T.E. Heidmann S. Seiwert S.D. Microbiol. Mol. Biol. Rev. 1997; 61: 105-120Crossref PubMed Scopus (140) Google Scholar, 8Simpson L. Thiemann O.H. Savill N.J. Alfonzo J.D. Maslov D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6986-6993Crossref PubMed Scopus (119) Google Scholar). Minicircles in most trypanosomatids species are heterogeneous in sequence. However, a few structural and sequence motifs are conserved in all the minicircles within networks of a given species as well as in minicircles of different trypanosomatid species. These include two short sequences that are associated with the process of replication initiation, which are located 70–100 nucleotides apart in the minicircle molecule; they are the dodecameric sequence GGGGTTGGTGTA, designated the universal minicircle sequence (UMS) and the hexameric sequence ACGCCC. These sequences have been mapped to the sites of the replication origins of the minicircle light (L) and heavy (H) strands, respectively (for review, see Refs. 1Ray D.S. Plasmid. 1987; 17: 177-190Crossref PubMed Scopus (45) Google Scholar, 5Shlomai J. Curr. Mol. Med. 2004; 4: 623-647Crossref PubMed Scopus (117) Google Scholar). Comparison of the complete minicircle sequences of several species of trypanosomatids reveals that minicircles from different trypanosomatids contain either one (Trypanosoma brucei GenBank™ accession number M15323; Leishmania major, GenBank™ accession number Z32845), two (C. fasciculata, GenBank™ accession number M19266), or three (Trypanosoma cruzi, GenBank™ accession number X56188) copies of the conserved origin region. The two conserved origin regions in C. fasciculata kDNA minicircles are designated OriA and OriB. Each of these origin regions includes the two conserved origin sequences. According to the currently accepted model for kDNA replication, only one of the origins is active during minicircle replication, and the active origin is randomly selected in each replication cycle (9Birkenmeyer L. Ray D.S. J. Biol. Chem. 1986; 261: 2362-2368Abstract Full Text PDF PubMed Google Scholar). Unlike the replication of mitochondrial DNA in other eukaryotic cells, which takes place throughout the cell cycle, kDNA replicates only once, during S phase of the cell cycle, approximately in parallel with the replication of the nuclear DNA (10Woodward R. Gull K. J. Cell Sci. 1990; 95: 49-57Crossref PubMed Google Scholar). According to the current model for kDNA replication, minicircles are released during S-phase from the center of the network by the decatenation activity of a type II DNA topoisomerase and are translocated to the kineto-flagellar zone, located between the kDNA network and the flagellum basal body (11Drew M.E. Englund P.T. J. Cell Biol. 2001; 153: 735-744Crossref PubMed Scopus (59) Google Scholar). Each minicircle is an individual replicon that replicates unidirectionally in a semi-discontinuous mechanism, forming two gapped and nicked progeny molecules (12Birkenmeyer L. Sugisaki H. Ray D.S. J. Biol. Chem. 1987; 262: 2384-2392Abstract Full Text PDF PubMed Google Scholar). Minicircles are then transferred onto two antipodal sites, flanking the kDNA disk, in which primer removal, repair of the Okazaki fragment gaps, and reattachment of the progeny minicircles to the network occurs. The final gap-filling and sealing of the topologically linked minicircles take place before the network division (13Kitchin P.A. Klein V.A. Englund P.T. J. Biol. Chem. 1985; 260: 3844-3851Abstract Full Text PDF PubMed Google Scholar). Several of the proteins involved in the replication of the kDNA network have been identified, including the origin-binding protein, designated UMS-binding protein (UMSBP). UMSBP has been purified from C. fasciculata, and its encoding gene and genomic locus were cloned and analyzed (14Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 17Tzfati Y. Shlomai J. Mol. Biochem. Parasitol. 1998; 94: 137-141Crossref PubMed Scopus (13) Google Scholar). Genes encoding for homologous proteins have been identified in other trypanosomatid species (18Hertz-Fowler C. Peacock C.S. Wood V. Aslett M. Kerhornou A. Mooney P. Tivey A. Berriman M. Hall N. Rutherford K. Parkhill J. Ivens A.C. Rajandream M.A. Barrell B. Nucleic Acids Res. 2004; 32: 339-343Crossref PubMed Google Scholar) (supplemental Table 1) The protein binds specifically to the two conserved sequences located at the minicircle replication origins, the UMS dodecamer and a 14-mer sequence (H14) containing the conserved hexamer core and 8 flanking nucleotides, in their single-stranded conformation (14Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 15Abeliovich H. Tzfati Y. Shlomai J. Mol. Cell. Biol. 1993; 13: 7766-7773Crossref PubMed Scopus (31) Google Scholar, 19Abu-Elneel K. Kapeller I. Shlomai J. J. Biol. Chem. 1999; 274: 13419-13426Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The 13.7-kDa protein contains five tandemly arranged CCHC-type zinc-knuckle motifs. This motif forms a compact zinc finger that has been associated with the binding of single-stranded nucleic acids (20Katz R.A. Jentoft J.E. BioEssays. 1989; 11: 176-181Crossref PubMed Scopus (29) Google Scholar, 22Rajavashisth T.B. Taylor A.K. Andalibi A. Svenson K.L. Lusis A.J. Science. 1989; 245: 640-643Crossref PubMed Scopus (201) Google Scholar). We have previously reported on the effect of redox on the binding of UMSBP to the origin sequence as well as on the protein oligomerization and suggested that redox potential may play a major role in the regulation of UMSBP action at the replication origin (23Onn I. Milman-Shtepel N. Shlomai J. Eukaryot. Cell. 2004; 3: 277-287Crossref PubMed Scopus (52) Google Scholar). Immunolocalization of UMSBP within the kineto-plast has revealed two distinct protein foci at the kineto-flagellar zone, near the suggested minicircle replication site (24Abu-Elneel K. Robinson D.R. Drew M.E. Englund P.T. Shlomai J. J. Cell Biol. 2001; 153: 725-734Crossref PubMed Scopus (58) Google Scholar). As a single-stranded DNA-binding protein, an intriguing question is, How does UMSBP bind the native origin in the duplex kDNA minicircle? This question had been addressed in earlier studies, demonstrating that the minicircle origin region is distorted and adopts a single-strand conformation (25Avrahami D. Tzfati Y. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10511-10515Crossref PubMed Scopus (24) Google Scholar). Yet no direct evidence has been provided in these studies for the binding of the full-length native origin, including both the L-strand and H-strand initiation sites, by UMSBP, and biochemical and structural analyses of this intriguing nucleoprotein complex have not yet been described. In this study we present evidence for the interaction of UMSBP with kDNA networks in vivo. We further describe the interactions of UMSBP with the sequences conserved at the native minicircle replication origin and show that UMSBP binding induces a significant conformational change in the DNA structure at the native minicircle replication origin. Preparation of UMSBP—Cloning of the C. fasciculata UMSBP gene open reading frame (15Abeliovich H. Tzfati Y. Shlomai J. Mol. Cell. Biol. 1993; 13: 7766-7773Crossref PubMed Scopus (31) Google Scholar) and the preparation of pure recombinant UMSBP were conducted as we have previously described (23Onn I. Milman-Shtepel N. Shlomai J. Eukaryot. Cell. 2004; 3: 277-287Crossref PubMed Scopus (52) Google Scholar). In Vivo Cross-linking and Purification of Kinetoplast Nucleoprotein Complexes—700 ml of C. fasciculata cell culture were grown to logarithmic phase (4.4 × 107 cells/ml) as described elsewhere (14Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 19Abu-Elneel K. Kapeller I. Shlomai J. J. Biol. Chem. 1999; 274: 13419-13426Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Cross-linking of kinetoplasts and their partial purification were carried out following the procedure of Xu and Ray (26Xu C. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1786-1789Crossref PubMed Scopus (64) Google Scholar) with modifications as follows. Formaldehyde (Sigma) was added to a final concentration of 0.75%, and the cells were incubated for 10 min at 37 °C with shaking followed by chilling in ice water. Cells were harvested in a Sorvall GS-3 rotor at 5000 rpm for 10 min at 4 °C and washed once with phosphate-buffered saline and once in NET buffer (10 mm Tris-Cl pH 8.0, 100 mm NaCl, 100 mm EDTA). Cells were then resuspended to final density of 1.25 × 109 cells/ml in NET buffer containing 3% sarkosyl and heated for 10 min at 65 °C. The cell lysate was fractionated through a series of three step gradients as described by Xu and Ray (26Xu C. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1786-1789Crossref PubMed Scopus (64) Google Scholar) as follows. Each 12.0-ml lysate was loaded on the top of a step gradient containing a layer of 23 ml of 20% sucrose in TNES buffer (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1% Sarkosyl) and a bottom layer of 3 ml of 20% sucrose in TNES, containing NaI to yield a density of 1.36 g/ml, and was centrifuged for 5 min at 10,000 rpm in a Beckman SW28 rotor at 25 °C. The bottom 7.6 ml was discarded, and the step-gradient was refilled from the bottom with 5 ml of 20% sucrose in TNES, 3 ml of 20% sucrose, NaI solution (1.36 g/ml), and 3 ml of saturated NaI in TNE (50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1 mm EDTA) and was centrifuged at 20,000 rpm for 10 min in a Beckman SW28 rotor at 25 °C. The bottom 10 ml of the gradient was fractionated into 10 1.0-ml fractions. To monitor their content of kDNA, samples withdrawn from the gradient fractions were electrophoresed in a 1% agarose gel and stained with ethidium bromide. The DNA-containing fractions (5Shlomai J. Curr. Mol. Med. 2004; 4: 623-647Crossref PubMed Scopus (117) Google Scholar, 8Simpson L. Thiemann O.H. Savill N.J. Alfonzo J.D. Maslov D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6986-6993Crossref PubMed Scopus (119) Google Scholar) were pooled, diluted with equal volume of TNES, and centrifuged in an Eppendorf 5417R at 14,000 rpm and 4 °C for 1 h. The pellets, containing kDNA networks, were resuspended in 18 ml of TNES. Each 9.0-ml suspension of DNA was re-centrifuged as described above through a step gradient containing (from top to bottom) 23 ml of 20% sucrose, 3 ml of 20% sucrose, NaI (1.36 g/ml) in TNES, and 3 ml of saturated NaI solution in TNE. The bottom 10 ml of the gradient was fractionated, and the fractions containing kDNA were diluted and centrifuged as described above. The nucleoprotein pellets were resuspended in 200 μl of TNES and loaded onto a 5.0 ml of sucrose/NaI linear gradient at the range of 10–30% sucrose, saturated NaI in 50 mm Tris-Cl, pH 8.0, 100 mm NaCl. The gradients were centrifuged at 20,000 rpm for 20 min in a Beckman SW55.5 rotor at 25 °C. 20 fractions of 250 μl were collected from the bottom of the tubes. Samples withdrawn from each fraction were analyzed for their kDNA content by electrophoresis in ethidium bromide-stained agarose gel followed by Southern blot hybridization analysis using a radioactively labeled minicircle probe following the published procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 9.31-9.58Google Scholar). Two of the DNA-containing fractions were randomly selected for further analysis. Each fraction was divided into halves. One-half was incubated overnight at 65 °C to reverse the formaldehyde cross-linking of the nucleoprotein complex, and the other was incubated overnight at 4 °C. The fractions were then added to loading buffer and incubated at 37 °C for 30 min and then electrophoresed in 16.5% SDS-PAGE as described below along with protein markers and recombinant UMSBP. Gels were analyzed by Western blot analysis using anti-UMSBP antibodies and Envision+ System HRP (DakoCytonation) secondary antibodies by ECL as described below. Immunoprecipitation of Nucleoprotein Complexes in Vitro—OriA region of the minicircle was amplified by PCR, using as template either a C. fasciculata kDNA minicircle or a plasmid containing a full-length minicircle (linearized by cleavage at its XhoI site). All the sets of primers (A-I) used for preparation of wild-type (WT) and mutated origin region in this work are described in supplemental Table 2. The DNA fragments were biotinylated using 5′-biotinylated primers. PCR amplification was carried out using Failsafe PCR kit (Epicenter Technologies). PCR products were purified using PCR cleaning column (Qiagen). PCR using primers set A (supplemental Table 2) yielded a 313-bp fragment (positions 319–631 (28Sugisaki H. Ray D.S. Mol. Biochem. Parasitol. 1987; 23: 253-263Crossref PubMed Scopus (35) Google Scholar)) that includes the OriA. Mutated OriA sequences were generated in two steps by PCR amplification using in the first step a 313-bp fragment of kDNA minicircle as the DNA template and two sets of mutated primers for each mutant, primers set B and C (supplemental Table 2), yielding two fragments with an overlap (underlined) at the H14 site. In the second step the fragments described above were used as templates with primers set A, yielding a 313-bp fragment that includes a mutated H14 (positions 396–409) and WT UMS sequence. In the first step primers set D and E yielded two fragments with an overlap (underlined, supplemental Table 2) at the UMS site. In the second step the fragments described above were used as template with the primers set A (supplemental Table 2), yielding a 313-bp fragment that included a WT H14 and mutated UMS sequences (positions 489–500). The template for the generation of a double mutant, mutated in both H14 and UMS was the PCR product of the 313-bp fragment that includes a WT H14 and mutated UMS. The two steps of PCR amplification were conducted as in the production of the mutated H14, as described above. PCR products produced in the first and second steps of the preparation of all mutants and the WT were purified by electrophoresis in agarose gel followed by their extraction using a gel extraction preparation (QIAquick, Qiagen). Binding of UMSBP to the double-stranded origin fragment was assayed in a 10-μl reaction mixture containing 150 ng (0.75 pmol) of the origin-containing PCR-amplified DNA fragments, 25 mm Tris-Cl, pH 7.5, 5 mm dithiothreitol, 2 mm MgCl2, and 1 mg/ml bovine serum albumin. Reactions started by the addition of UMSBP (7.5 pmol) as indicated were incubated at 30 °C for 60 min. The reaction mixtures were cooled on ice for 10 min, and nucleoprotein complexes were cross-linked by UV light (UV Stratalinker 1800, Stratagene) irradiated at 306 mJ/cm2 on ice. Reactions were adjusted to 200 μl by the addition of IP buffer (10 mm Tris-Cl, pH 8.0, 500 mm NaCl, 1% Nonidet P-40, and 2 mm EDTA) and added to protein A beads preincubated for 2 h with rabbit anti-UMSBP antibodies. The reactions were incubated overnight at 4 °C with constant rotation. The beads were washed 3 times, with 0.7 ml of phosphate-buffered saline (PBS; 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.4) containing 1% Nonidet P-40 and then twice with 0.7 ml of PBS. The beads were resuspended in 10 mm Tris-Cl, pH 7.5, 0.1 mm EDTA containing 1% sarkosyl and 2 mg/ml proteinase K (Roche Applied Science) and incubated for 2 h at 37 °C followed by overnight incubation at 60 °C. The DNA was then electrophoresed in 2% agarose gel, vacuum-blotted onto a Hybond-N+ membrane (Amersham Biosciences), and analyzed by Southern blot hybridization using a minicircle DNA probe. Quantification of radioactive signals was conducted using BAS2000 (Fuji) phosphorimaging. Preparation of Minicircle Origin (OriA) and Mutated OriA Fragments for Binding and Electrophoretic Mobility Shift Analysis (EMSA) Analyses—The minicircle fragment containing the OriA region of C. fasciculata kDNA (see above) was PCR-amplified using Failsafe PCR kit (Epicenter Technologies). PCR primer set F (supplemental Table 2) was used for the preparation of the WT OriA-containing fragment yielding a 142-bp fragment (positions 377–518). Mutated OriA sequences were generated by PCR amplification using primers that include mutated origins. The 142-bp fragment containing OriA served as template. Primers sets (supplementary Table 2) were as follows. Set G yielded a 144-bp fragment (positions 377–520) that included a mutated H14 and WT UMS sequences; set H yielded a 144-bp fragment (positions 377–520) that included a WT H14 and a mutated UMS sequence, and set I yielded a 144-bp fragment (positions 377–520) that included a double mutant (a mutated H14 and a mutated UMS sequence). The WT and mutated PCR products were purified by electrophoresis in agarose gel followed by their extraction from the gel using a gel extraction preparation (QIAquick). The primers used for the preparation of ligands for micrococcal nuclease digestion of nucleoprotein complexes were 5′-32P-labeled using [γ-32P]ATP and T4 polynucleotide kinase (MBI Fermentas). EMSA—Analyses were carried out as described previously (14Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 16Tzfati Y. Abeliovich H. Avrahami D. Shlomai J. J. Biol. Chem. 1995; 270: 21339-21345Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Samples of UMSBP as indicated were incubated in the 10-μl binding reaction mixture containing 25 mm Tris-Cl, pH 7.5, 2 mm MgCl2, 5 mm dithiothreitol, 10% glycerol, 1 μg/μl bovine serum albumin, 0.25 μg of poly(dI-dC)·poly(dI-dC), and 25 fmol of 5′-32P-labeled DNA ligand as indicated. Reactions were incubated at 30 °C for 15 min as indicated. The reaction mixtures were cooled on ice for 10 min, and nucleoprotein complexes were cross-linked by UV light as described above. The products were loaded onto an 8% native polyacrylamide gel (1:29, bisacrylamide/acrylamide) in TAE buffer (6.7 mm Tris acetate, 3.3 mm sodium acetate, 1 mm EDTA, pH 7.5). Electrophoresis was conducted at 2–4 °C and 16 V/cm for 3 h. Surface Plasmon Resonance (SPR) Analysis—SPR studies were conducted using a BIAcore 3000 at the BIAcore unit, the Hebrew University of Jerusalem. A 5′-biotinylated 313 bp DNA was prepared using primer set A (supplementary Table 2: the primer 5′-CCATGGGTGTGTTTGTGTTG TTCTGG-3′ is 5′-biotinylated). Biotinylated DNA was immobilized to an SA sensor chip (BIAcore), as recommended by the manufacturer (60–500 RU). DNA binding activity of the proteins was measured by injection of 100–300 nm UMSBP into the flow channel, using an empty flow-channel as background. Kinetic analysis was performed by automated injection of various protein concentrations (30 μl/min, 3-min association time and 3-min dissociation time) in 10 mm Hepes pH 8.0, 150 mm NaCl, 5 mm dithiothreitol, 2 mm MgCl2. No mass transfer was detected under these conditions. Binding constants were calculated by BIAevaluation 3.1 program using a heterogeneous ligand binding model. Micrococcal Nuclease Assay—Samples of UMSBP as indicated were added to a reaction mixture containing 0.25 pmol of 5′-32P-labeled 313-bp minicircle DNA fragment (positions 319–631, GenBank™ accession number M19266), 25 mm Tris-Cl, pH 7.5, 2 mm MgCl2, 5 mm CaCl2, and 20 mm dithiothreitol. The reaction was incubated for 60 min at 30 °C followed by the addition of 1 unit of micrococcal nuclease (MBI Fermentas) for 10 min at 37 °C. The reaction was stopped by the addition of 25 mm EGTA, and the reaction products were loaded onto a 6% polyacrylamide sequencing gel (1:19, bisacrylamide/acrylamide) along with a sequencing reaction of minicircle DNA using the ddNTPs procedure (U. S. Biochemical Corp.) and a 5′-CCATGGGTGTGTTTGTGTTG-3′ primer. Thermodynamic Analysis—Structural properties of the C. fasciculata minicircle sequence (GenBank™ accession number M19266) were analyzed for DNA curvature according to Bolshoy et al. (29Bolshoy A. McNamara P. Harrington R.E. Trifonov E.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2312-2316Crossref PubMed Scopus (437) Google Scholar) and propeller-twist, as a measure of helix rigidity, as described by El Hassan and Calladine (30el Calladine C.R. Hassan M.A. J. Mol. Biol. 1996; 259: 95-103Crossref PubMed Scopus (186) Google Scholar). Window size was 100 bp, and slide was 10 bp. Analysis of Proteins by SDS-PAGE—Protein samples in loading buffer containing 50 mm Tris-Cl, pH 6.85, 4% SDS, 3.5% β-mercaptoethanol, 10% (v/v) glycerol, and 10 mm EDTA were incubated at 37 °C for 30 min and loaded onto a 16.5% Tris-Tricine SDS-polyacrylamide gel (31Schagger H. Von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10457) Google Scholar) along with protein size markers (Rainbow pre-stained low molecular weight, Amersham Biosciences). Upper electrophoresis buffer was 0.1 m Tris-Tricine, pH 8.25, containing 0.1% SDS; lower buffer was 0.2 m Tris-Cl, pH 8.9. Western Blot Analysis—Protein samples were analyzed by SDS-PAGE electrophoresis, as described above. Protein bands were transferred onto a Protran BA85 cellulose nitrate membrane (Schleicher & Schuell). Membranes were blocked by incubation for 30 min in 5% skim dry milk (Difco) in phosphate-buffered saline containing 0.1% (v/v) Tween 20 and probed for 90 min with anti-UMSBP antibodies that were raised in rabbit. Membranes were probed for 45 min with a 1:13,000 dilution horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (DacoCytonation) followed by ECL detection as recommended by the manufacturer (Amersham Biosciences). UMSBP Interacts in Vivo with kDNA Networks—UMSBP was immunolocalized within the kinetoplast to two discrete foci located at the kineto-flagellar zone (24Abu-Elneel K. Robinson D.R. Drew M.E. Englund P.T. Shlomai J. J. Cell Biol. 2001; 153: 725-734Crossref PubMed Scopus (58) Google Scholar) at the site implicated with kDNA minicircle replication initiation (11Drew M.E. Englund P.T. J. Cell Biol. 2001; 153: 735-744Crossref PubMed Scopus (59) Google Scholar). Moreover, immunofluorescence analysis of synchronized C. fasciculata culture revealed a higher abundance of UMSBP at this site during S-phase, in correlation with the progress of kDNA replication. However, whereas binding of UMSBP to single-stranded origin sequences has been previously demonstrated in DNA binding assays in vitro, its interaction with native kDNA networks in vivo has not yet been challenged. This question was addressed here using an in vivo protein-DNA cross-linking approach following a procedure modified from Xu and Ray (26Xu C. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1786-1789Crossref PubMed Scopus (64) Google Scholar). Lysates prepared from cross-linked cells were fractionated by centrifugation through a series of sucrose/NaI stepwise gradients (26Xu C. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1786-1789Crossref PubMed Scopus (64) Google Scholar), and pooled kDNA-containing fractions obtained were fractionated by a linear sucrose/NaI gradient. Two kDNA-containing fractions (Fig. 1A) from the final linear sucrose/NaI gradient were monitored by SDS-PAGE analysis followed by Western blot analysis using anti-UMSBP antibodies either with or without heat reversal of the nucleoprotein cross-linking (Fig. 1B). In both kDNA-containing fractions, reversal of the formaldehyde cross-linking by heating at 65 °C overnight (26Xu C. Ray D.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1786-1789Crossref PubMed Scopus (64) Google Scholar) resulted in the release of UMSBP as indicated by the subsequent SDS-PAGE and Western blot analysis, whereas only traces of protein were released from the untreated complexes (Fig. 1B). Under the denaturing and reducing conditions used in this analysis the majority of released protein migrated in the gel as monomers. The minor slower migrating bands observed (arrowheads, Fig. 1B) may represent traces of higher oligomeric forms of UMSBP, generated under the cross-linking conditions, that resisted the denaturing and reducing electrophoresis conditions. These results indicate that UMSBP interacts in vivo with kDNA networks. Interactions of UMSBP with free kDNA minicircles are undetectable by the method employed here, since the purification of the large kDNA-protein complexes in the sucrose/NaI gradients apparently excludes unlinked free minicircles. UMSBP Binds in Vitro Double-stranded DNA Fragments Carrying the Minicircle Replication Origin—We have previously shown that UMSBP is a single-stranded DNA-binding protein that binds specifically the 12-mer UMS and the 14-mer sequence containing the core hexamer (H14) in their single-stranded conformation (14Tzfati Y. Abeliovich H. Kapeller I. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6891-6895Crossref PubMed Scopus (52) Google Scholar, 16Tzfati Y. Abeliovich H. Avrahami D. Shlomai J. J. Biol. Chem. 1995; 270: 21339-21345Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 19Abu-Elneel K. Kapeller I. Shlomai J. J. Biol. Chem. 1999; 274: 13419-13426Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In binding competition analyses duplex native kDNA minicircles, either free or topologically linked, competed efficiently with a radioactively labeled single-stranded UMS on the binding of UMSBP (25Avrahami D. Tzfati Y. Shlomai J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10511-10515Crossref PubMed Scopus (24) Google Scholar). However, no direct evidence has yet been reported, demonstrating the binding of UMSBP to the native minicircle origin, which includes both its UMS and H14 sequences in a duplex kDNA conformation. To examine the binding of UMSBP at the origin and its dependence upon the presence of its UMS and/or H14 binding sites" @default.
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