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• W2030483729 abstract "Using a combined PCR-gel retardation assay, the preferred recognition sequence of the Streptomycesinitiator protein DnaA was determined. The protein showed a preference toward DNA containing two Escherichia coli-like DnaA boxes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas the consensus sequence of the DnaA boxes found in theStreptomyces oriC region is TTGTCCACA). In quantitative band shift experiments, the kinetics of the StreptomycesDnaA-DnaA box interaction was characterized. The DnaA protein can form dimers while binding to a single DnaA box; dimer formation is mediated by the domain III of the protein, and the dissociation constant of this process was between 35 and 115 nm. Streptomycesinitiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance separating the DnaA boxes, domain I (or I and II) is responsible. The cooperativity constant is moderate and is in the range of 20–110. Using a combined PCR-gel retardation assay, the preferred recognition sequence of the Streptomycesinitiator protein DnaA was determined. The protein showed a preference toward DNA containing two Escherichia coli-like DnaA boxes in a head-to-head arrangement (consensus sequence TTATCCACA, whereas the consensus sequence of the DnaA boxes found in theStreptomyces oriC region is TTGTCCACA). In quantitative band shift experiments, the kinetics of the StreptomycesDnaA-DnaA box interaction was characterized. The DnaA protein can form dimers while binding to a single DnaA box; dimer formation is mediated by the domain III of the protein, and the dissociation constant of this process was between 35 and 115 nm. Streptomycesinitiator protein DnaA interacts in a cooperative manner with DNA containing multiple binding sites. For the cooperativity effect, which seems to be independent of the distance separating the DnaA boxes, domain I (or I and II) is responsible. The cooperativity constant is moderate and is in the range of 20–110. base pair(s) electrophoretic mobility shift assay polymerase chain reaction surface plasmon resonance The initiator protein DnaA plays an essential role in the initiation of bacterial chromosome replication. The interaction of DnaA protein with its chromosomal origin (oriC) is best understood in Escherichia coli. The E. coli DnaA protein (52 kDa) binds to five nonpalindromic, nonamer sequences, the DnaA boxes. Binding of 10–20 DnaA monomers promotes a local unwinding of an adjacent AT-rich region. The unwound region provides an entry site for the DnaB/DnaC helicase complex followed by other proteins required to form a replication fork (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman and Co., New York1992Google Scholar, 2Skarstad K. Boye E. Biochim. Biophys. Acta. 1994; 1217: 111-130Crossref PubMed Scopus (168) Google Scholar, 3Messer W. Weigel C. Neidhardt F.C. Curtiss III, R. Ingraham J. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 1601-1779Google Scholar, 4Kaguni J.M. Mol. Cells. 1997; 7: 145-157PubMed Google Scholar). Apart from its primary function as a replisome organizer, the DnaA protein acts as a transcription factor that represses or activates several genes or terminates transcription, depending on the location and arrangement of DnaA boxes (5Messer W. Weigel C. Mol. Microbiol. 1997; 24: 1-6Crossref PubMed Scopus (123) Google Scholar). Both functions of the DnaA protein, replisome organizer and transcription factor, are mediated by its interaction with target DNA. The DNA binding domain of E. coli DnaA has been localized in the 94 C-terminal amino acids. A potential helix-loop-helix motif has been reported within this part of the protein (6Roth A. Messer W. EMBO J. 1995; 14: 2106-2111Crossref PubMed Scopus (122) Google Scholar). However, because x-ray high resolution structure analysis is not yet available for DnaA proteins, the detailed interaction with the DNA is still poorly understood. The consensus sequence of the E. coli DnaA box differs depending on the method used for its evaluation. The most stringent definition for the DnaA box sequence comes from a determination of binding constants: 5′-TT(A/T)TNCACA-3′ (7Schaper S. Messer W. J. Biol. Chem. 1995; 270: 17622-17626Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). T2, T4, T7′, and T9′were found to be directly involved in DNA-protein interaction (8Speck C. Weigel C. Messer W. Nucleic Acids Res. 1997; 25: 3242-3247Crossref PubMed Scopus (23) Google Scholar).E. coli DnaA does not dimerize in solution and interacts with a single DnaA box as a monomer, as measured by gel retardation (7Schaper S. Messer W. J. Biol. Chem. 1995; 270: 17622-17626Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) and by surface plasmon resonance (9Speck C. Weigel C. Messer W. EMBO J. 1999; 18: 6169-6176Crossref PubMed Scopus (153) Google Scholar). However, binding to DnaA boxes that differ from the stringent consensus sequence by one or two base pairs requires two such boxes and an interaction of DnaA proteins bound to them (9Speck C. Weigel C. Messer W. EMBO J. 1999; 18: 6169-6176Crossref PubMed Scopus (153) Google Scholar). DnaA binds ATP and ADP with high affinity. Both forms of DnaA protein, ATP-DnaA and ADP-DnaA, recognize DNA in a similar fashion; however, only ATP-DnaA is active in the DNA replication process. Recently, it has been shown that E. coli ATP-DnaA protein recognizes also a hexamer sequence, the ATP-DnaA box 5′-AGATCT-3′ or close match of it (9Speck C. Weigel C. Messer W. EMBO J. 1999; 18: 6169-6176Crossref PubMed Scopus (153) Google Scholar). Streptomycetes (Gram-positive soil bacteria) differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large (8-megabase pair), linear and GC-rich (about 72%) chromosome (10Kutzner H.J. Starr M.P. Stolp H. Trüper H.G. Balows A. Schlegel H.G. The Prokaryotes. Springer, Berlin1981: 2028-2090Google Scholar, 11Lin Y.S. Kieser H.M. Hopwood D.A. Chen C.W. Mol. Microbiol. 1993; 10: 923-933Crossref PubMed Scopus (229) Google Scholar). Recent discoveries suggest that replication of the linear chromosome of Streptomyces coelicolor A3(2) proceeds bidirectionally from the centrally located oriC region toward the ends of the chromosome (12Musialowski M.S. Flett F. Scott G.B. Hobbs G. Smith C.P. Oliver S.G. J. Bacteriol. 1994; 176: 5123-5125Crossref PubMed Google Scholar). The key elements of initiation of the Streptomyceschromosome replication, oriC region and DnaA protein, show higher complexity than those of E. coli. TheStreptomyces oriC region contains numerous DnaA boxes, which are grouped into two clusters (13Zakrzewska-Czerwiñska J. Schrempf H. J. Bacteriol. 1992; 174: 2688-2693Crossref PubMed Google Scholar, 14Jakimowicz D. Majka J. Messer W. Speck C. Fernandez M. Martin M.C. Sanchez J. Schauwecker F. Keller U. Schrempf H. Zakrzewska-Czerwiñska J. Microbiology. 1998; 144: 1281-1290Crossref PubMed Scopus (61) Google Scholar). The StreptomycesDnaA protein consists, like all other DnaA proteins, of four domains. In contrast to the other bacteria, the Streptomyces DnaA protein is larger (70–73 kDa), since it comprises an additional stretch (∼230 amino acids) of predominantly acidic or hydrophobic amino acids within domain II. The residues lower the isoelectric point of the entire Streptomyces DnaA protein (pI = 5.7) (15Majka J. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Bacteriol. 1997; 179: 2426-2432Crossref PubMed Google Scholar,16Majka J. Jakimowicz D. Messer W. Schrempf H. Lisowski M. Zakrzewska-Czerwiñska J. Eur. J. Biochem. 1999; 260: 325-335Crossref PubMed Scopus (30) Google Scholar). As it was shown for the E. coli and Bacillus subtilis DnaA proteins, domain III and the C-terminal part (domain IV) of the Streptomyces DnaA protein are responsible for binding of ATP and DNA, respectively (15Majka J. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Bacteriol. 1997; 179: 2426-2432Crossref PubMed Google Scholar, 17Majka J. Characterization of the Streptomyces lividans initiator protein DnaA, Ph.D. thesis. Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland1997Google Scholar). The consensus sequence of the Streptomyces DnaA box in oriC is 5′-TTGTCCACA-3′, which differs at the third position (A′G) from theE. coli DnaA box (13Zakrzewska-Czerwiñska J. Schrempf H. J. Bacteriol. 1992; 174: 2688-2693Crossref PubMed Google Scholar). In contrast to E. coli, the Streptomyces DnaA protein can form a dimer when binding to a single DnaA box. Recently, it has been shown that the domains I and III are independently involved in dimerization of theStreptomyces lividans DnaA protein molecules. The interaction of Streptomyces DnaA protein with two DnaA boxes is cooperative and accompanied by strong DNA bending (16Majka J. Jakimowicz D. Messer W. Schrempf H. Lisowski M. Zakrzewska-Czerwiñska J. Eur. J. Biochem. 1999; 260: 325-335Crossref PubMed Scopus (30) Google Scholar, 18Jakimowicz D. Majka J. Konopa G. Wgrzyn G. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Mol. Biol. 2000; 298: 351-364Crossref PubMed Scopus (30) Google Scholar). However, we do not know the contribution of the different domains of theStreptomyces DnaA protein to cooperative binding. In this work, we apply a combined PCR-EMSA1 technique (19Pollock R. Treisman R. Nucleic Acids Res. 1990; 18: 6197-6204Crossref PubMed Scopus (290) Google Scholar) to elucidate DNA sequence requirements for Streptomyces DnaA protein binding. Using EMSA, we define details of the binding of theStreptomyces wild type DnaA and its truncated forms to DNA. We reveal that the domain I participates in cooperative DnaA protein-DNA interactions. Quantitative analysis of gel mobility shifts allowed us to determine binding constants for dimerization and cooperative DNA protein interactions. In addition, we have varied the spacing between two DnaA boxes and examined the consequences on dimerization and cooperative binding of DnaA protein to these boxes. The His-tagged wild type DnaA proteins ofS. lividans and its truncated mutants DnaA(III-IV) comprising the domain III and the DNA binding domain were purified on a Ni2+-nitrilotriacetic acid-agarose column (Qiagen) as described earlier (15Majka J. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Bacteriol. 1997; 179: 2426-2432Crossref PubMed Google Scholar). The DNA binding domain of the DnaA protein DnaA(BD) was expressed as a C-terminal glutathioneS-transferase fusion and purified using glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) followed by factor Xa cleavage as described earlier (16Majka J. Jakimowicz D. Messer W. Schrempf H. Lisowski M. Zakrzewska-Czerwiñska J. Eur. J. Biochem. 1999; 260: 325-335Crossref PubMed Scopus (30) Google Scholar). Oligonucleotides were chemically synthesized and purified by high performance liquid chromatography. For the kinetic analysis of DnaA protein binding complementary nucleotides were annealed by heating at 95 °C for 10 min and gradually cooling to room temperature. The oligonucleotides were end-labeled with [γ-32P]ATP and T4 polynucleotide kinase and purified from 5%nondenaturing polyacrylamide gels. The recognition motif of the DnaA protein was determined using several cycles of amplification and selection, essentially as described previously (19Pollock R. Treisman R. Nucleic Acids Res. 1990; 18: 6197-6204Crossref PubMed Scopus (290) Google Scholar). The library of 58-bp oligonucleotides for the first selection cycle was prepared by PCR using template oligonucleotides: 5′-GGCGGATCCTCGACTAGCGN20GCTACGAGCTGAGCTCGCG-3′ and the primer pair 5′-GGCGGATCCTCGACTAGCG-3′ and 5′-CGCGAGTCTAGCTCGTAGC-3′ (restriction sitesBamHI and SacI are in italic type). The amplification reaction was carried out in 100 μl using 1 pmol of template oligonucleotide (1 pmol corresponds to the number of all possible combinations of 20 degenerated bases) and 100 pmol of each primer for 20 cycles, with each cycle consisting of 15 s at 96 °C, 30 s at 60 °C, and 30 s at 72 °C. Double-stranded oligonucleotides were separated on 3%agarose gels, electroeluted for 1 h to TBE buffer, and purified by phenol-chloroform extraction and glycogen-ethanol precipitation. The oligonucleotides were 5′-end-labeled with [γ-32P]ATP and T4 polynucleotide kinase and then incubated with DnaA protein in concentrations of 5 or 50 nm in binding buffer (20 mm HEPES-KOH, pH 8.0, 5 mm magnesium acetate, 1 mm Na2EDTA, 4 mm dithiothreitol, 0.2%Triton X-100, 5 mg/ml bovine serum albumin, and 100 μm ATP) The reaction was carried out in 20 μl for 30 min at room temperature in the presence of competitor, poly(dI-dC), at a concentration of 2.5 μg/μl. The complexes were separated on 5%nondenaturing polyacrylamide gels in 0.25× TBE. The bands corresponding to DNA-protein complexes were excised, and the DNA was eluted into gel elution buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 5 mm MgCl2) for 3 h at 60 °C and recovered by phenol-chloroform extraction followed by glycogen-ethanol precipitation. The DNA was then amplified by PCR at conditions described above and subjected to five additional selection cycles of binding and amplification. The subsequent cycles differed only in the amount of competitor added to binding reactions (for cycles 2–6, we used 5, 7.5, 20, 50, and 100 μg/μl, respectively). Selected oligonucleotides after the fourth, fifth, and sixth round were digested with BamHI and SacI and cloned into a pUC19 vector. The DNA from independent clones was recovered using a Spin Miniprep Kit (Qiagen) and subjected to sequencing using32P-5′-end-labeled “−47” primer (New England Biolabs) and the Thermo Sequenase cycle sequencing kit (Amersham Pharmacia Biotech). For binding assays, 5′-32P-end-labeled DNA (TableI; ≤0.01 nm) was incubated with different amounts of the DnaA proteins (concentration range indicated in the legend to Fig. 2) in the presence of a competitor (poly(dI-dC), 10 ng/μl) at 20 °C for 30 min in binding buffer (20 mm HEPES-KOH, pH 8.0, 5 mm magnesium acetate, 1 mm Na2EDTA, 4 mm dithiothreitol, 0.2%Triton X-100, 5 mg/ml bovine serum albumin, and 100 μm ATP). The free DNA and complexes were separated by electrophoresis on 5%native polyacrylamide gels, prerun for 1 h (0.25× TBE, 6 V/cm, 20 °C). Following electrophoresis, the radioactive gel was dried and analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA). The variation in experimental data was evaluated by repeating each experiment three or four times. Different protein titration experiments showed variations up to 15%. Equilibrium constants were determined using the modified statistical mechanical model (20Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar) supplemented with the protein dimerization module. For the DNA substrate containing a single binding site A, the concentration of each species in the gel was expressed by the following equations (model 1),Table IOligonucleotides used in the gel retardation experimentsThe black arrow shows the strong Streptomyces DnaA box; the white arrow shows the E. coli DnaA box R1/R4; the grey arrow shows the box2 found by the in vitro DNA binding sites selection assay; and the crossed out arrow shows the scrambled DnaA box. Open table in a new tab The black arrow shows the strong Streptomyces DnaA box; the white arrow shows the E. coli DnaA box R1/R4; the grey arrow shows the box2 found by the in vitro DNA binding sites selection assay; and the crossed out arrow shows the scrambled DnaA box. S=1/Z1Equation 1 SL=kAL/Z1Equation 2 SL2=kAkDiL2/Z1Equation 3 Z1=1+kAL+kAkDiL2Equation 4 where S, SL, andSL2 represent concentrations of free DNA, monomer, and dimer respectively; L is protein (ligand) concentration; kA is the microscopic equilibrium affinity constant for binding site A, and kDi is the affinity constant describing the dimerization process. For the DNA containing binding sites A and B, the concentrations of the species presented in the gel are described by the following equations (model 2), S=1/Z2Equation 5 SL=(kA+kB)L/Z2Equation 6 SL2=(kDi[kA+kB]+kAkBkAB)L2/Z2Equation 7 Z2=1+(kA+kB)L+(kDi(kA+kB)+kAkBkAB)L2Equation 8 where kB is the intrinsic affinity constant for the binding site B, and kAB is the cooperativity constant describing interaction of protein molecules occupying both binding sites. Experimental data were simultaneously fitted to the equations using Scientist® for WindowsTM software (MicroMath). The consensus sequence of the DnaA box identified within theStreptomyces oriC region is 5′-TTGTCCACA-3′. TheStreptomyces oriC region contains a higher number of the DnaA boxes (19 boxes) than the E. coli oriC region (five boxes). A DNase I footprint of the oriC region with DnaA showed protection of all boxes. However, when analyzed individually by surface plasmon resonance (SPR), only some DnaA boxes were specifically recognized by the DnaA protein (14Jakimowicz D. Majka J. Messer W. Speck C. Fernandez M. Martin M.C. Sanchez J. Schauwecker F. Keller U. Schrempf H. Zakrzewska-Czerwiñska J. Microbiology. 1998; 144: 1281-1290Crossref PubMed Scopus (61) Google Scholar, 15Majka J. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Bacteriol. 1997; 179: 2426-2432Crossref PubMed Google Scholar). To characterize in detail the recognition properties of the Streptomyces DnaA protein, we applied a binding site selection technique based on the combined EMSA and polymerase chain reactions (Fig.1 A; Ref. 19Pollock R. Treisman R. Nucleic Acids Res. 1990; 18: 6197-6204Crossref PubMed Scopus (290) Google Scholar). The purified DnaA protein was incubated with a substrate pool of double-stranded oligonucleotides, in which a random 20-bp region was flanked by defined 19-bp sequences containing restriction sites to facilitate subsequent cloning. The protein-DNA complexes were separated from the unbound DNA on a 5%polyacrylamide nondenaturing gel. Only one protein-bound fraction was visible. The DNA recovered from the complex was amplified by PCR with the pair of primers complementary to the defined sequences of the oligonucleotides (Fig. 1 B) and used as a substrate for the renewed binding assay. In the third selection cycle, we observed a second retarded band with a higher electrophoretic mobility than the previous one. Here, the bands with lower and higher electrophoretic mobility are called a “dimer” (Fig. 1 C, ** complex) and a “monomer” (Fig.1 C, * complex), respectively. The DNA from both bands was recovered and used independently in the next selection cycles. The alignments of the selected oligonucleotides after the fourth, fifth, and sixth cycle are shown in Tables II andIII.Table IISelected DNA binding sites for DnaA (“dimer” band)Box B1Box B2Cycle 420×tagcgGCAGTTATCCACATGTGGATgctac10×tagcgGTTATCCACATGTGTATGCAgctac3×tagcgGAAGGGTTGTCCACATGTGTgctac2×gtagcCTAGGTTATCCACATGTGTAcgcta2×tagcgTTCAGAGTTATCCACCTGTGgctac2×tagcgGCGATGTTATCCACATGTGGgctac2×tagcgTTGTGCACATGTGTATGATTgctactagcgGCAGTTATCCACATGAGGATgctactagcgAGGTTTATCCACATGTTGATgctactagcgGCAGTTATCCACATGTGGAGgctactagcgGGTTGCTTATCCACATGTGGgctactagcgTTGCCTTATGCACATGTGCAgctactagcgTTCCTGTTATGCACATGTGTgctacgtagcCTAAGTTATCCACATGTGTGcgctatagcgGTGTTATGCACACTGTGAGgctactagcgCTGGTTTTCCACATGTGGATgctactagcgGGTTTTCCACAGGTGTGCATgctactagcgGACCTTGTCCACATGTGTATgctactagcgGGGGTTGTCCACATGTGTACgctactagcgCACTGTGCACATGTGCGTAgctactagcgGCTCGGATATCCACATGTGTgctactagcgTGTGAGTTATCCCCATGTGGgctactagcgGTATCCACAATGTGTATGCAgctacgtcgcAATCATACACATGTGCACAcgctatagcgTGTAGTAGTCCACATGTGAGgctactagcgTAATGTTGTGCACAGGTGTCgctactagcgGTTTCGGTTGTGCACATGTgctactagcgTGACCGTCCACATGTGCGTGgctacgtcgcCATCGTCCACAACCACACACcgctagtcgcCACAATATCCACAAGCGTGAcgctagtcgcCTAGGTATCCACAAGTTGAAcgctagtcgcCCACAACCTTATCCCCAGCGcgctagtcgcCGACGACTTCCACAGCAGAAcgctaAGATConsensus sequenceNNGTT TCCACATGTGg ctNNgTgcCycle 537×tagcgGCAGTTATCCACATGTGGATgctac12×tagcgGTTATCCACATGTGTATGCAgctac4×gtagcCTAgGTTATCCACATGTGTAcgctatagcgGTAGTTATCCACATGTGGATgctactagcgGCGGTTATCCACATGTGGATgctactagcgGTCATCCACATGTGTATGCAgctacgtagcCTAAGTTATCCACATGTGTAcgctagtagcCGAGTTATGCACAAGTGGATcgctatagcgCTAGTTATCCACAACCCGAAgctacAGTAConsensus sequenceC GTTATCCACATGTG ATGC CGTagCycle 629×tagcgGCAGTTATCCACATGTGGATgctac3×tagcgGTTATCCACATGTGTATGCAgctactagcgGTTATCCACATGTGCATGCAgctactagcgGTAGTTATCCACATGTGGATgctactagcgGCAGTTATCCACAGGTGGATgctactagcgGCAGTTACCCACATGTGGATgctacgtagcCAACTTATCCACAGGCACGGcgctaConsensus sequenceCAGTTATCCACATGTGGATGCTACThe sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50–85%. Nucleotides that are represented at less than 15%were considered as not significant at the indicated position. Open table in a new tab Table IIISelected DNA binding sites for DnaA (“monomer” band)Box B1Box B2Cycle 420×tagcgGCAGTTATCCACATGTGGATgctac2×tagcgGTTATCCACATGTGTATGCAgctactagcgGCAGTTATCCACATGTGGACgctactagcgGCGATGTTATCCACATGTGGgctactagcgGCGATATTATCCACATGTGGgctacgtagcCATGGTTATCCACATGTGTAcgctatagcgGTAGTTATCCACAAGTGGATgctac8×gtagcCAACTTATCCACAGGCACGGcgcta2×gtagcCAAGTTATCCACAACCCGGAcgcta2×gtagcCCACACAGTTATCCACAGGCcgctagtagcCCAGGACTTATCCACAGGTAcgctatagcgTACGGGTTATCCACAACTAGgctacgtagcCGAGTTATCCACAGAATGAAcgctagtagcACAGAGTTATCCACAGGTTAcgctatagcGTAGTTATCCACAGATTGGCtacC GT TGGATGC TACConsensus sequenceATTATCCACAGa cG cacGgcG CtACycle 58×tagcgGCAGTTATCCACATGTGGATgctac2×tagcgGTTATCCACATGTGTATGCAgctactagcgGCAGTTATCCACATGTGGGTgctac19×gtcgcCAACTTATCCACAGGCACGGcgcta2×tagcGTAGTTATCCACAGGTTGGCtac2×gtcgcCTAGGTTATCCACAGGCGTAcgctatagcgTAGTTATCCACAGATTGgctacgtcgcCTAGTTATCCACAAGCCCTAcgctatagcgTACTGTTATCCACAGGCTGGgctacgtcgcCTAGTTATCCACAGGCCGTAcgctagtagcCTAGTTATCCACAGGTCGTAcgctagtagcACAGAGTTATCCACAGGTTAcgctagtagcCAAGTTATCCACAACCCGTAcgctagtagcCAAGTTATCCACAACCCGGAcgctagtagcTCCAGGTTATCCACAGCGCTcgctagtagcCCCCAAGGTTATCCACAGCTcgctaA Gg cacggcg ctaConsensus sequenceATTATCCACAGc Ct tggttgc tacCycle 6tagcgGCAGTTATCCACAGGTGGATgctac25×gtagcCAACTTATCCACAGGCACGGcgcta4×gtagcCCACACAGTTATCCACAGGCcgcta3×tagcgTAGTTATCCACAGATTGgctac2×gtagcCAAGTTATCCACAACCCGGAcgcta2×gtagcACAGAGTTATCCACAGGTTAcgctagtagcCAACTTATCCACAGGCAGGGcgctagtagcCAACTTATCCACAGACACGGcgctatagcgGTTATCCACAGGTGTATGCAgctacgtagcCAACTTATCCACAGGTACGGcgctagtagcCTAGTTATCCACAGGTCGTGcgctagtagcGCAACTTATCCACAAGTGGAcgctacgactAGCGAAGTTATCCACAGggctaccC cConsensus sequenceAATTATCCACAGGA GGCG CTAgt gThe sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50–85%. Nucleotides that are represented at less than 15%were considered as not significant at the indicated position. Open table in a new tab The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50–85%. Nucleotides that are represented at less than 15%were considered as not significant at the indicated position. The sequence of cloned binding sites for DnaA, derived after the fourth, fifth, and sixth selection cycles, were aligned for maximum match to the canonical DnaA box definition. The region of 20 originally random nucleotides is shown in uppercase letters, whereas nucleotides from adjacent primer regions are indicated by lowercase letters (only five bases from each side of the random region are shown). Letters in boldface type match the DnaA box consensus sequence. Letters in italic type indicate the lower strand of the DnaA box. For the consensus sequence, letters in boldface represent nucleotides present more than 85%. Uppercase letters indicate nucleotides appearing in the frequency range 50–85%. Nucleotides that are represented at less than 15%were considered as not significant at the indicated position. The sequences selected from the “dimer” band after four rounds reveal remarkable features: 63 out of the 67 oligonucleotides analyzed carry two DnaA boxes, one complete 9-bp DnaA box, the “box 1,” and one incomplete DnaA box, the “box 2” (“box” as defined by sequence). The incomplete box 2 consists of 4–7 bp (3′-part of the DnaA box). Both boxes face each other and are adjacent (except for a few oligonucleotides). 80%of the boxes 1 exhibit the E. coli type of DnaA box (T or A at the third position), and only 20%of them exhibit the Streptomyces type (G at the third position). During the next two rounds, the selection progressively narrowed the spectrum of observed oligonucleotide sequences; after six rounds the box 1 is exclusively represented by the E. coliDnaA box R1/R4 (5′-TTATCCACA-3′), and the partial box 2 contains in nearly all cases the essential T nucleotides at the 7′- and 9′-positions. More than half of the oligonucleotides selected from the monomer band after four rounds contained also two boxes. However, the monomer band had only appeared after the third round of the selection (Fig.1 C), and therefore it still contained traces of the oligonucleotides from the dimer band that were subsequently amplified. After the next two selection rounds, only one out of the 44 oligonucleotides contained two boxes. All of the boxes 1 from the monomer band exhibit the E. coli type DnaA box (R1/R4). The data show that under the conditions of the assay, theStreptomyces DnaA protein interacts only with DnaA boxes; no other consensus sequences (e.g. the newly identified 6-bp ATP-DnaA box (9Speck C. Weigel C. Messer W. EMBO J. 1999; 18: 6169-6176Crossref PubMed Scopus (153) Google Scholar)) have been found. The results, presented in Tables IIand III, show that DnaA protein from Streptomyces possesses a higher affinity toward DnaA boxes from E. coli than toward those from its own oriC region. Binding of the DnaA protein as a dimer apparently requires “head-to-head” orientation of the boxes. Upstream of the promoter region of the Streptomyces dnaA gene are two closely spaced DnaA boxes: a strong one (with the preferredStreptomyces sequence: 5′-TTGTCCACA-3′) and a weak one (5′-TTGTCCCCA-3′) in head-to-head arrangement with 3 bp in between (21Zakrzewska-Czerwiñska J. Nardmann J. Schrempf H. Mol. Gen. Genet. 1994; 242: 440-447Crossref PubMed Scopus (20) Google Scholar). Interaction of the DnaA protein with these boxes creates an autoregulatory circuit similar to that known for the E. coli dnaA gene (21Zakrzewska-Czerwiñska J. Nardmann J. Schrempf H. Mol. Gen. Genet. 1994; 242: 440-447Crossref PubMed Scopus (20) Google Scholar). Recently, we have shown that binding of the DnaA protein to both DnaA boxes exhibits a cooperative character (16Majka J. Jakimowicz D. Messer W. Schrempf H. Lisowski M. Zakrzewska-Czerwiñska J. Eur. J. Biochem. 1999; 260: 325-335Crossref PubMed Scopus (30) Google Scholar). Domains I and III independently participate in the dimerization of the DnaA protein molecules (18Jakimowicz D. Majka J. Konopa G. Wgrzyn G. Messer W. Schrempf H. Zakrzewska-Czerwiñska J. J. Mol. Biol. 2000; 298: 351-364Crossref PubMed Scopus (30) Google Scholar). To evaluate in detail the kinetics of binding of the DnaA protein to DnaA boxes from the dnaA gene promoter region, we applied gel mobility shift assays to determine the binding constants for cooperativity as well as for dimerization (20Senear D.F. Brenowitz M. J. Biol. Chem. 1991; 266: 13661-13671Abstract Full Text PDF PubMed Google Scholar). This assay permits the quantitative analysis of the individual protein-DNA complexes. In our gel retardation experiments, we used the wild-type DnaA protein and its truncated forms, the DnaA(III-IV) lacking the two N-terminal domains (I and II) and the DnaA(BD) containing only the DNA binding domain IV. As a prerequisite for the kinetic studies, two DNA substrates containing either a single strong DnaA box, 1s-0 (the weak DnaA box was scrambled), or two DnaA boxes, 2s , derived from the promoter region of the dnaA gene were designed in such a way that the DnaA box(es) i" @default.
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• W2030483729 title "Sequence Recognition, Cooperative Interaction, and Dimerization of the Initiator Protein DnaA of Streptomyces" @default.
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