FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017904568> ?p ?o ?g. }

• W2017904568 endingPage "50471" @default.
• W2017904568 startingPage "50465" @default.
• W2017904568 abstract "PriB is one of the Escherichia coli φX-type primosome proteins that are required for assembly of the primosome, a mobile multi-enzyme complex responsible for the initiation of DNA replication. Here we report the crystal structure of the E. coli PriB at 2.1 Å resolution by multi-wavelength anomalous diffraction using a mercury derivative. The polypeptide chain of PriB is structurally similar to that of single-stranded DNA-binding protein (SSB). However, the biological unit of PriB is a dimer, not a homotetramer like SSB. Electrophoretic mobility shift assays demonstrated that PriB binds single-stranded DNA and single-stranded RNA with comparable affinity. We also show that PriB binds single-stranded DNA with certain base preferences. Based on the PriB structural information and biochemical studies, we propose that the potential tetramer formation surface and several other regions of PriB may participate in protein-protein interaction during DNA replication. These findings may illuminate the role of PriB in φX-type primosome assembly. PriB is one of the Escherichia coli φX-type primosome proteins that are required for assembly of the primosome, a mobile multi-enzyme complex responsible for the initiation of DNA replication. Here we report the crystal structure of the E. coli PriB at 2.1 Å resolution by multi-wavelength anomalous diffraction using a mercury derivative. The polypeptide chain of PriB is structurally similar to that of single-stranded DNA-binding protein (SSB). However, the biological unit of PriB is a dimer, not a homotetramer like SSB. Electrophoretic mobility shift assays demonstrated that PriB binds single-stranded DNA and single-stranded RNA with comparable affinity. We also show that PriB binds single-stranded DNA with certain base preferences. Based on the PriB structural information and biochemical studies, we propose that the potential tetramer formation surface and several other regions of PriB may participate in protein-protein interaction during DNA replication. These findings may illuminate the role of PriB in φX-type primosome assembly. A bacterial primosome is a multi-enzyme complex that participates in DNA replication. It travels along the lagging strand template, unwinds the duplex DNA, and primes the Okazaki fragments that are required for replication fork progression (1Schekman R. Weiner A. Kornberg A. Science. 1974; 186: 987-993Crossref PubMed Scopus (107) Google Scholar, 2Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman & Co., New York1992: 275-306Google Scholar). Escherichia coli φX-type primosome was originally discovered as an essential component for the replication of bacteriophage φX174 and ColE1-type plasmids (3Wickner S. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 4120-4124Crossref PubMed Scopus (103) Google Scholar, 4Minden J.S. Marians K.J. J. Biol. Chem. 1985; 260: 9316-9325Abstract Full Text PDF PubMed Google Scholar). It is now clear that the primary role of the φX-type primosome is to restart the stalled replication fork at oriC after encountering DNA damage (5Marians K.J. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 39-67Crossref PubMed Google Scholar, 6Marians K.J. Trends Biochem. Sci. 2000; 25: 185-189Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Sandler S.J. Marians K.J. J. Bacteriol. 2000; 182: 9-13Crossref PubMed Scopus (176) Google Scholar, 8Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (871) Google Scholar). During replication restart, in vitro evidence suggests that the φX-type primosome is located at the D-loop, a three-way-junction DNA structure that is an intermediate in recombination repair, and that PriA initiates the primosome assembly at the D-loop (9McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (165) Google Scholar, 10Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 11Liu J. Xu L. Sandler S.J. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3552-3555Crossref PubMed Scopus (116) Google Scholar, 12Xu L. Marians K.J. Mol. Cell. 2003; 11: 817-826Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Phage φX174 takes advantage of the unique substrate preference of PriA and has evolved a pas 1The abbreviations used are: pas, primosome assembly site; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; SSB, single-stranded DNA-binding protein; EcoSSB, Escherichia coli SSB; HsmtSSB, human mitochondrial SSB; pHMB, sodium p-hydroxymer-curibenzoate; MAD, multi-wavelength anomalous diffraction; NEM, N-ethylmaleimide.1The abbreviations used are: pas, primosome assembly site; ssDNA, single-stranded DNA; ssRNA, single-stranded RNA; SSB, single-stranded DNA-binding protein; EcoSSB, Escherichia coli SSB; HsmtSSB, human mitochondrial SSB; pHMB, sodium p-hydroxymer-curibenzoate; MAD, multi-wavelength anomalous diffraction; NEM, N-ethylmaleimide. (primosome assembly site) sequence that diverts the primosomes from host chromosomal replication to phage DNA production (5Marians K.J. Prog. Nucleic Acids Res. Mol. Biol. 1999; 63: 39-67Crossref PubMed Google Scholar). PriB is one of the seven essential proteins (PriA, PriB, PriC, DnaB, DnaC, DnaT, and DnaG) for the assembly of the φX-type primosome for phage φX174 replication (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar, 14Allen Jr., G.C. Kornberg A. J. Biol. Chem. 1991; 266: 11610-11613Abstract Full Text PDF PubMed Google Scholar, 15Zavitz K.H. DiGate R.J. Marians K.J. J. Biol. Chem. 1991; 266: 13988-13995Abstract Full Text PDF PubMed Google Scholar). The assembly of a primosome on the φX174 viral DNA, as described in the following sentences, is an ordered process (16Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15642-15648Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). (i) PriA recognizes and binds to the pas. (ii) PriB joins PriA to form a PriA-PriB-pas DNA complex. (iii) DnaT then joins this complex to form a triprotein complex on the pas DNA; and (iv) DnaB is then transferred from a DnaB-DnaC complex to the PriA-PriB-DnaT-pas DNA complex to form a preprimosome assembly that consists of PriA, PriB, DnaT, and DnaB on the DNA. Finally, DnaG adds to this complex by interacting with DnaB and completing the primosome assembly (17Tougu K. Peng H. Marians K.J. J. Biol. Chem. 1994; 269: 4675-4682Abstract Full Text PDF PubMed Google Scholar). Although in vitro studies have demonstrated that PriC was not required for the stable preprimosome assembly on the 304-nucleotide pas DNA sequence (16Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15642-15648Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), it is a component of the bound preprimosome that was isolated from the full-length φX174 viral DNA (18Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The PriA-directed assembly of the φX-type primosome on the D-loop has been shown to be similar to that on the pas sequence of φX174 viral DNA (10Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). During the formation of the preprimosome assembly, PriB interacts with and stabilizes the PriA-DNA complex (10Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 16Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15642-15648Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), facilitating the complex formation between PriA and DnaT (19Liu J. Nurse P. Marians K.J. J. Biol. Chem. 1996; 271: 15656-15661Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). PriB was formerly known as the “n protein” because it can be inactivated by treatment with N-ethylmaleimide (NEM) (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar). In vitro experiments indicate that the modified PriB protein decreased the primosomal replication activity of phage φX174 (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar). Cells harboring priB mutations display defects in PriA-dependent replication restart (20Sandler S.J. Marians K.J. Zavitz K.H. Coutu J. Parent M.A. Clark A.J. Mol. Microbiol. 1999; 34: 91-101Crossref PubMed Scopus (75) Google Scholar, 21Sandler S.J. McCool J.D. Do T.T. Johansen R.U. Mol. Microbiol. 2001; 41: 697-704Crossref PubMed Scopus (49) Google Scholar, 22Flores M.J. Ehrlich S.D. Michel B. Mol. Microbiol. 2002; 44: 783-792Crossref PubMed Scopus (37) Google Scholar) and ColE1-type plasmid replication (23Berges H. Oreglia J. Joseph-Liauzun E. Fayet O. J. Bacteriol. 1997; 179: 956-958Crossref PubMed Google Scholar, 24Harinarayanan R. Gowrishankar J. Genetics. 2004; 166: 1165-1176Crossref PubMed Scopus (12) Google Scholar). PriB forms dimers in solution (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar, 14Allen Jr., G.C. Kornberg A. J. Biol. Chem. 1991; 266: 11610-11613Abstract Full Text PDF PubMed Google Scholar), and each preprimosome may contain two PriB dimers (18Ng J.Y. Marians K.J. J. Biol. Chem. 1996; 271: 15649-15655Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). PriB can bind single-stranded DNA (ssDNA) in the presence or absence of single-stranded DNA-binding protein (SSB) in vitro (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar, 25Allen Jr., G.C. Kornberg A. J. Biol. Chem. 1993; 268: 19204-19209Abstract Full Text PDF PubMed Google Scholar). PriB is generally considered to be a structural component of the φX-type primosome. However, the function of this protein in primosome assembly is not fully understood at the molecular level. Recent studies on sequence comparisons and operon organization analyses have shown that PriB evolved from SSB via gene duplication with subsequent rapid sequence diversification (26Ponomarev V.A. Makarova K.S. Aravind L. Koonin E.V. J. Mol. Microbiol. Biotechnol. 2003; 5: 225-229Crossref PubMed Scopus (21) Google Scholar). SSB has long been known for its importance in DNA replication (27Meyer R.R. Laine P.S. Microbiol. Rev. 1990; 54: 342-380Crossref PubMed Google Scholar). It raises an interesting question as to how PriB participates in DNA replication in a fashion different from that of its ancestor. In this article we present the crystal structure of the E. coli PriB at 2.1 Å resolution. Based on its structural features as well as results from biochemical and mutation studies, we discuss the potential role of PriB in φX-type primosome assembly. Cloning, Protein Expression, and Purification—The wild-type priB gene was amplified by PCR from E. coli strain K12. PCR primers were designed to incorporate unique EcoRI and SalI restriction sites, permitting the insertion of the amplified gene into the pET21b vector (Novagen) for protein expression in E. coli. To facilitate the PCR experiment, Gly-103 in the recombinant PriB was substituted with valine by the design of the reverse primer. The resultant plasmid, pET21b-PriB, encodes a recombinant PriB protein fused with an N-terminal T7 tag (MASMTGGQQMGRDPNSL) and a C-terminal His tag (KLAAALEHHHHHH). The T7 tag facilitates the detection of the recombinant protein in primosome reconstitution experiments, whereas the His tag is useful for recombinant protein purification. The coding sequence of this plasmid was further confirmed by DNA sequencing. E. coli BL21(DE3) cells were used as host cells for expression of the recombinant PriB proteins. The His-tagged recombinant proteins were purified with a Co2+ chelating column (TALON®, Clontech) according to the manufacturer's recommendation. The eluted protein was dialyzed against the crystallization buffer (0.1 m NaCl and 20 mm Tris-HCl at pH 7.9) and concentrated to 10 mg ml-1 for crystallization. Crystallization and Data Collection—Both native and sodium p-hydroxymercuribenzoate (pHMB) derivative crystals of PriB were grown using the hanging drop vapor diffusion method at 25 °C. Native crystals grew within 2 weeks after mixing 1 μl of protein solution (10 mg ml-1 in 0.1 m NaCl and 20 mm Tris-HCl at pH 7.9) with 0.5 μl of 1 m l-cysteine and 1 μl of reservoir solution containing 25% (w/v) polyethylene glycol 5000 monomethyl ester in 50 mm sodium acetate at pH 5.7. The crystals belong to space group P212121 with cell dimensions a = 39.32 Å, b = 57.36 Å, and c = 97.02 Å and diffract to 2.1 Å. There are two molecules per asymmetric unit. The native data set was collected on a Rigaku R-AXIS IV++ image-plate detector (Rigaku, MSC) using copper Kα radiation from a Rigaku RU-300 rotating anode x-ray generator operated at 50 kV and 80 mA under cryogenic conditions. After an extensive heavy atom derivative search, one useful mercury derivative was obtained by the co-crystallization method under a slightly different condition. Briefly, 2 μl of protein solution was mixed with 0.5 μlof20mm pHMB, 0.5 μlof1 m l-cysteine, and 1 μl of reservoir solution containing 25% polyethylene glycol 4000 in 100 mm sodium acetate at pH 5.5 and 100 mm Tris-HCl at pH 8.5. The pHMB derivative crystals appeared after 3 days. They share the same space group and similar cell dimensions with the native crystals but diffract only to 2.5 Å. The structure of PriB was determined using multi-wavelength anomalous diffraction (MAD) phasing (28Hendrickson W.A. Science. 1991; 254: 51-58Crossref PubMed Scopus (1016) Google Scholar) applied to mercury. The MAD data collection was conducted at the R-AXIS-IV++ imaging plate using a synchrotron radiation x-ray source at Beamline 17B2 of the National Synchrotron Radiation Research Center (NSRRC) in Taiwan. The MAD data were collected at four wavelengths under cryogenic conditions. Two energies were chosen near the absorption peak and edge of mercury, 1.00826 and 1.00933 Å, corresponding to the maximum f″ and the minimum f′, respectively. Two remote energies were selected as reference wavelengths in 0.992097 and 1.02493 Å, respectively. Data integration and scaling were performed using the HKL package (29Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38555) Google Scholar) (Table I).Table IData collection and refinement statisticsData setNativepHMB derivativeλ1 (Peak)λ2 (Edge)λ3 (Remote 1)λ4 (Remote 2)Wavelength (Å)1.54181.008261.009330.9920971.02493Resolution (Å)2.12.52.52.52.5Space groupP212121P212121P212121P212121P212121Redundancy3.62.44.62.54.6Completeness (%)99.9 (99.6)aValues in parenthesis indicate those for the highest resolution shell97.1 (98.7)99.8 (100.0)98.5 (99.3)99.6 (98.8)Average I/σ(I)14.1 (2.2)15.0 (5.9)15.8 (8.1)14.6 (3.9)18.4 (9.7)RmergebRmerge (I) = ΣhΣI|Ii - I|ΣhΣI I, where I is the mean intensity of the i observations of reflection h (%)8.3 (56.1)7.7 (19.4)7.8 (18.6)9.7 (38.3)7.0 (15.0)RefinementResolution limit (Å)24.0-2.1R (%)20.6Rfree (%)26.3Number of atomsProtein1704Cysteine21Water134Average B-factor (Å2)All atoms39.9Protein39.5Cysteine44.4Water44.6R.m.s.cR.m.s. is root mean square deviationBond length (Å)0.006Bond angle (°)1.3a Values in parenthesis indicate those for the highest resolution shellb Rmerge (I) = ΣhΣI|Ii - I|ΣhΣI I, where I is the mean intensity of the i observations of reflection hc R.m.s. is root mean square Open table in a new tab Structure Determination and Refinement—SOLVE (30Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3219) Google Scholar) was used to locate the mercury sites and generate the initial MAD phases at 3 Å resolution. The initial phases were extended and further improved to 2.5 Å by RESOLVE (31Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 38-44Crossref PubMed Scopus (593) Google Scholar). XtalView (32McRee D.E. J. Struct. Biol. 1999; 125: 156-165Crossref PubMed Scopus (2021) Google Scholar) was used to examine electron density maps and molecular models. The native data set (2.1 Å) was used for further refinement using CNS (33Brűnger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. GrosseKunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16963) Google Scholar). During refinement, non-crystallographic symmetry neglecting the loop regions was imposed initially but released after two monomers in the asymmetric unit were built. The final model of PriB contains all residues for monomer A and monomer B. The residues preceding position 1 of each monomer are from the T7 tag, and the residues after position 104 of monomer A are from the His tag. The current model has an R-factor of 20.6% for all reflections above 2 σ between 24 and 2.1 Å resolution and an Rfree of 26.3% using 5% randomly distributed reflections. The Ramachandran plot has no violation of accepted backbone torsion angles. The statistical data are shown in Table I. The atomic coordinates of PriB have been deposited in the Protein Data Bank under accession code 1V1Q. Preparation of 32P-Labeled Probes—DNA oligonucleotides 35 bases long, (dT)35 and (dA)35, were purchased from Integrated DNA Technologies, whereas an RNA oligonucleotide, U35, of the same length was obtained from Dharmacon. RNA I and RNA II carrying a T7 promoter sequence were synthesized using an in vitro transcription system (Riboprobe®, Promega). These probes were 5′-end labeled with T4 polynucleotide kinase (Promega) and [γ-32P]ATP (6000 Ci mmol-1; PerkinElmer Life Sciences). The probes were purified on denaturing polyacrylamide gel and resuspended in TE buffer (0.1 mm EDTA and 10 mm Tris-HCl at pH 8). The concentrations of the 32P-labeled probes were estimated with isotope counting. Electrophoretic Mobility Shift Assay—To analyze the formation of ssDNA- or single-stranded RNA (ssRNA)-PriB complexes, 0.01 pmol of 32P-labeled (dT)35, (dA)35, or U35 was mixed with various amounts of recombinant PriB protein in a 10-μl-reaction mixture containing 40 mg ml-1 bovine serum albumin and 100 mm HEPES at pH 7 for 30 min at 25 °C. The resulting samples were resolved on a native 12% polyacrylamide gel (30:1 acrylamide/bisacrylamide) at 4 °C in TBE buffer (89 mm Tris borate and 1 mm EDTA) and visualized by autoradiography. Complexed and free DNA bands were scanned and quantified. The PriB-ssDNA binding dissociation constants (Kd) were estimated from the protein concentration that binds 50% of the input DNA (34Fairall L. Buttinelli M. Panetta G. Travers A. Buckle M. DNA-Protein Interactions: A Practical Approach. Oxford University Press, New York2000: 65-74Google Scholar). In this report, each Kd is calculated as the average of at least three measurements ± S.D. To assess the binding ability of PriB to RNA I or RNA II, the reactions were carried out by mixing 1 pmol of 32P-labeled RNA I or RNA II with various amounts of recombinant PriB protein in a 10-μl reaction volume for 30 min at 4 °C. The resulting complexes were resolved on a native 10% polyacrylamide gel at 4 °C and visualized by autoradiography. The Overall Structure of the PriB Monomer—The PriB crystal belongs to the orthorhombic space group P212121 with two molecules in an asymmetric unit. Two identical polypeptide chains form a dimer with a non-crystallographic 2-fold symmetry (Fig. 1A). A PriB monomer is a single domain protein (residues 1 to 104) topologically identical to the well characterized OB (oligonucleotide-binding) fold (35Murzin A.G. EMBO J. 1993; 12: 861-867Crossref PubMed Scopus (772) Google Scholar). Briefly, the PriB monomer structure has two pleated β-sheets capped by a small α-helix located between the third and the fourth strands to form a β-barrel (Fig. 1B). The core of the β-barrel is filled with hydrophobic residues. Between the anti-parallel strands PriB has three β-hairpin loops termed L12 (residues 20–24), L23 (residues 37–44), and L45 (residues 81–88) that protrude from the central core. Structural comparison of the two monomers in the asymmetric unit reveals that the most significant structural variations between the monomers occur in the regions of the three loops (Fig. 1C). The C terminus (residues 101–104) is located at the tip of the β-barrel and protrudes from the main body of the barrel with considerable variation in its conformation as well. The root mean square deviation between the Cα positions (104 atoms in each monomer involved) in the two monomers is 2.54 Å. Neglecting the C termini and these loop regions decreases the root mean square deviation to 0.55 Å (79 atoms in each monomer involved). Despite the relatively low sequence identity with other single-stranded DNA-binding proteins (SSBs) (Fig. 1D), the PriB monomer displays high structural similarity to the ssDNA-binding domains of E. coli SSB (EcoSSB), human mitochondrial SSB (HsmtSSB), and Mycobacterium tuberculosis SSB (MtuSSB) (36Raghunathan S. Ricard C.S. Lohman T.M. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6652-6657Crossref PubMed Scopus (191) Google Scholar, 37Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (150) Google Scholar, 38Saikrishnan K. Jeyakanthan J. Venkatesh J. Acharya N. Sekar K. Varshney U. Vijayan M. J. Mol. Biol. 2003; 331: 385-393Crossref PubMed Scopus (59) Google Scholar). The tracings of the Cα atoms of these proteins are shown in Fig. 1C and demonstrate clearly that the globular core of these proteins has similar conformation. However, unlike other SSBs, the three loop regions of PriB are distinct in their conformation and orientation with respect to the core. Sequence alignment reveals two gaps that significantly shorten the L23 and L45 hairpin loops of PriB (Fig. 1D). L45 of EcoSSB has been shown to be involved in interface interactions between dimers for the one of the tetramer formations (36Raghunathan S. Ricard C.S. Lohman T.M. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6652-6657Crossref PubMed Scopus (191) Google Scholar). L45 of PriB, however, was not stabilized through crystal packing as shown in EcoSSB. Electron density for the tip of L45 is relatively weak and exhibited high atomic temperature factors. Architecture of the PriB Dimer—Both HsmtSSB and EcoSSB function as homotetramers in solution (39Curth U. Urbanke C. Greipel J. Gerberding H. Tiranti V. Zeviani M. Eur. J. Biochem. 1994; 221: 435-443Crossref PubMed Scopus (83) Google Scholar, 40Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (361) Google Scholar). In contrast to these SSBs, the PriB molecules form dimers in solution (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar, 14Allen Jr., G.C. Kornberg A. J. Biol. Chem. 1991; 266: 11610-11613Abstract Full Text PDF PubMed Google Scholar) and crystal (Fig. 1A). The two monomers in the PriB dimer are related by a non-crystallographic 2-fold axis that is similar to that of the EcoSSB or HsmtSSB dimer (Figs. 1A and 2). The dimeric interactions of PriB are fairly strong. The intermolecular contacts involve side chains and six short CO· · ·NH main chain H-bonds from β1a of each monomer, which build up the continuity of the three-stranded β-sheets of each subunit. Consequently, a six-stranded anti-parallel pleated sheet extends over the dimer. In addition to the interactions from the intermolecular anti-parallel strands, the arm region (Arg-34 to Trp-47; L23 and parts of the connecting β2 and β3) bends to one side of the paired monomer with intimate contacts involving van der Waals interactions and five hydrogen bonds (Fig. 1A). This close state structural feature is unique and has never been reported for crystal structures of SSBs, even for those that bind with ssDNA (37Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (150) Google Scholar, 40Raghunathan S. Kozlov A.G. Lohman T.M. Waksman G. Nat. Struct. Biol. 2000; 7: 648-652Crossref PubMed Scopus (361) Google Scholar, 41Matsumoto T. Morimoto Y. Shibata N. Kinebuchi T. Shimamoto N. Tsukihara T. Yasuoka N. J. Biochem. (Tokyo). 2000; 127: 329-335Crossref PubMed Scopus (52) Google Scholar). The PriB dimer is further stabilized by a symmetrical pair of intermolecular disulfide bridges formed by Cys-48 of one monomer to Cys-80 of the other monomer. These intermolecular interactions have not been observed in other SSBs. In addition to the disulfide bridges, side chains of Met-50 and Met-90 from both subunits form a methionine cluster (Fig. 1A) that locates between the two intermolecular disulfide bridges. Together, the methionine cluster and the disulfide bonds constitute the hydrophobic core of the extended β-barrel in the PriB dimer. Because of the close contact of the arm region of one subunit to the β-barrel of the other (Fig. 1A), the intermolecular interactions of PriB are much stronger than those of EcoSSB or HsmtSSB. The total surface area buried through dimerization in PriB is calculated to be ∼3545 Å2 as compared with that of 2335 and 2894 Å2 for EcoSSB and HsmtSSB, respectively. In addition, over two-thirds (69%) of the buried surface in PriB is contributed by hydrophobic interactions. In comparison, the percentage of the buried hydrophobic surface area is 52 and 54% in EcoSSB and HsmtSSB, respectively. PriB Fails to Form Homotetramers—PriB does not form a tetrameric structure in crystal (this work) or solution (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar, 14Allen Jr., G.C. Kornberg A. J. Biol. Chem. 1991; 266: 11610-11613Abstract Full Text PDF PubMed Google Scholar). In EcoSSB or HsmtSSB the homotetramer is a dimer of dimers with a 222 symmetry and has molecular 2-fold dyads along three intersecting axes (Fig. 2A). Two β-strands, β1a and β4, are important for the homotetramer formation (36Raghunathan S. Ricard C.S. Lohman T.M. Waksman G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6652-6657Crossref PubMed Scopus (191) Google Scholar, 37Yang C. Curth U. Urbanke C. Kang C. Nat. Struct. Biol. 1997; 4: 153-157Crossref PubMed Scopus (150) Google Scholar). Residues in β1a and β4 of PriB share only low sequence similarity to those of EcoSSB or HsmtSSB (Fig. 1D). In EcoSSB, a hydrophobic cluster formed by four symmetry-related isoleucine residues (Ile-9) stabilizes the six-stranded β-sheet-mediated tetramer interface (42Webster G. Genschel J. Curth U. Urbanke C. Kang C. Hilgenfeld R. FEBS Lett. 1997; 411: 313-316Crossref PubMed Scopus (63) Google Scholar). Similarly, four symmetric His-18 residues in HsmtSSB substitute for those isoleucine residues in EcoSSB. In PriB, however, the corresponding residue is Val-6. The low steric extent of the valine side chains probably cannot stabilize the potential tetramer interface (Fig. 2A). In addition, the charge distribution of PriB at the potential tetramer formation surface is considerably different from that in EcoSSB or HsmtSSB (Fig. 2B). In EcoSSB and HsmtSSB, a pair of charged residues (Lys-7 and Glu-80 in EcoSSB and Arg-16 and Glu-95 in HsmtSSB) form a cluster of intermolecular salt bridges at the tetramer formation surface and lock the orientation of subunits. In PriB, the corresponding Arg-4 and Glu-95/Glu-98 are unable to constitute a similar intermolecular salt bridge cluster such as that of EcoSSB or HsmtSSB. The abundance of negatively charge residues (Glu-58, Glu-95, and Glu-98) on the potential tetramer formation surface may also prevent homotetramer formation in PriB. Possible Roles of the Potential Tetramer Formation Surface of PriB—A priB triple mutant (G56E, H57N, and E58K) has been reported to be defective in the cellular replication of ColE1-related plasmids (23Berges H. Oreglia J. Joseph-Liauzun E. Fayet O. J. Bacteriol. 1997; 179: 956-958Crossref PubMed Google Scholar). Coincidentally, Glu-58 is located at the potential tetramer formation surface of PriB (Fig. 2B). Therefore, the negatively charged area containing Glu-58, Glu-95, and Glu-98 on the potential tetramer formation surface can possibly be a determinant for protein-protein interaction between PriB and other proteins during primosome assembly. Direct interaction between PriB and EcoSSB has been shown, and this protein complex retains ssDNA-binding ability (13Low R.L. Shlomai J. Kornberg A. J. Biol. Chem. 1982; 257: 6242-6250Abstract Full Text PDF PubMed Google Scholar). Interestingly, bacterial SSBs from different species can form cross-species heterotetramers through their six-stranded β-sheet surfaces (43De Vries J. Genschel J. Urbanke C. Thole H. Wackernagel W. Eur. J. Biochem. 1994; 224: 613-622Crossref PubMed Scopus (17) Google Scholar). This finding suggests that the similar six-stranded β-sheet surfaces of the PriB dimer could interact with an EcoSSB dimer. However, the formation of this heterotetramer has not been reported, and this possibility requires further investigations. Base Preference of Single-stranded DNA Binding of PriB—To assess the in vitro ssDNA binding ability of PriB, purified proteins were incubated with different amounts of 32P-labeled ssDNA in electrophoretic mobility shift assay-type experiments (Fig. 3, A and B). EcoSSB has two major ssDNA-binding modes that occlude 35 ± 2 and 65 ± 3 nucleotides per tetramer (44Lohman T.M. Overman L.B. J. Biol. Chem. 1985; 260: 3594-3603Abstract Full" @default.
• W2017904568 created "2016-06-24" @default.
• W2017904568 creator A5045892311 @default.
• W2017904568 creator A5062688152 @default.
• W2017904568 creator A5063532752 @default.
• W2017904568 creator A5065817705 @default.
• W2017904568 creator A5072353570 @default.
• W2017904568 creator A5073163299 @default.
• W2017904568 creator A5075927733 @default.
• W2017904568 date "2004-11-01" @default.
• W2017904568 modified "2023-10-07" @default.
• W2017904568 title "Crystal Structure of PriB, a Primosomal DNA Replication Protein of Escherichia coli" @default.
• W2017904568 cites W10876090 @default.
• W2017904568 cites W1504662341 @default.
• W2017904568 cites W1518548089 @default.
• W2017904568 cites W1522284441 @default.
• W2017904568 cites W1534892032 @default.
• W2017904568 cites W1537872391 @default.
• W2017904568 cites W1539796472 @default.
• W2017904568 cites W1558934363 @default.
• W2017904568 cites W1559132509 @default.
• W2017904568 cites W1560669282 @default.
• W2017904568 cites W1567167803 @default.
• W2017904568 cites W1570361288 @default.
• W2017904568 cites W1585812111 @default.
• W2017904568 cites W1806123628 @default.
• W2017904568 cites W1924027056 @default.
• W2017904568 cites W1965277349 @default.
• W2017904568 cites W1969156185 @default.
• W2017904568 cites W1977109739 @default.
• W2017904568 cites W1985188411 @default.
• W2017904568 cites W1985662030 @default.
• W2017904568 cites W1989840530 @default.
• W2017904568 cites W1991781564 @default.
• W2017904568 cites W1994920362 @default.
• W2017904568 cites W1995017064 @default.
• W2017904568 cites W2004392681 @default.
• W2017904568 cites W2007146285 @default.
• W2017904568 cites W2015840719 @default.
• W2017904568 cites W2019591778 @default.
• W2017904568 cites W2026307209 @default.
• W2017904568 cites W2027762453 @default.
• W2017904568 cites W2028231353 @default.
• W2017904568 cites W2044016235 @default.
• W2017904568 cites W2049098060 @default.
• W2017904568 cites W2065976083 @default.
• W2017904568 cites W2076469829 @default.
• W2017904568 cites W2077283568 @default.
• W2017904568 cites W2077652583 @default.
• W2017904568 cites W2079243965 @default.
• W2017904568 cites W2081765867 @default.
• W2017904568 cites W2091529967 @default.
• W2017904568 cites W2097766238 @default.
• W2017904568 cites W2102773702 @default.
• W2017904568 cites W2110730225 @default.
• W2017904568 cites W2121367329 @default.
• W2017904568 cites W2134141760 @default.
• W2017904568 cites W2135006530 @default.
• W2017904568 cites W2135839939 @default.
• W2017904568 cites W2140616356 @default.
• W2017904568 cites W2149730508 @default.
• W2017904568 cites W2154208108 @default.
• W2017904568 cites W2155885923 @default.
• W2017904568 cites W2158616999 @default.
• W2017904568 cites W2163884297 @default.
• W2017904568 cites W2165108940 @default.
• W2017904568 cites W4233022955 @default.
• W2017904568 cites W4293003482 @default.
• W2017904568 doi "https://doi.org/10.1074/jbc.m406773200" @default.
• W2017904568 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15383524" @default.
• W2017904568 hasPublicationYear "2004" @default.
• W2017904568 type Work @default.
• W2017904568 sameAs 2017904568 @default.
• W2017904568 citedByCount "42" @default.
• W2017904568 countsByYear W20179045682012 @default.
• W2017904568 countsByYear W20179045682013 @default.
• W2017904568 countsByYear W20179045682014 @default.
• W2017904568 countsByYear W20179045682017 @default.
• W2017904568 countsByYear W20179045682018 @default.
• W2017904568 countsByYear W20179045682019 @default.
• W2017904568 countsByYear W20179045682020 @default.
• W2017904568 countsByYear W20179045682021 @default.
• W2017904568 countsByYear W20179045682022 @default.
• W2017904568 countsByYear W20179045682023 @default.
• W2017904568 crossrefType "journal-article" @default.
• W2017904568 hasAuthorship W2017904568A5045892311 @default.
• W2017904568 hasAuthorship W2017904568A5062688152 @default.
• W2017904568 hasAuthorship W2017904568A5063532752 @default.
• W2017904568 hasAuthorship W2017904568A5065817705 @default.
• W2017904568 hasAuthorship W2017904568A5072353570 @default.
• W2017904568 hasAuthorship W2017904568A5073163299 @default.
• W2017904568 hasAuthorship W2017904568A5075927733 @default.
• W2017904568 hasBestOaLocation W20179045681 @default.
• W2017904568 hasConcept C104317684 @default.
• W2017904568 hasConcept C12554922 @default.
• W2017904568 hasConcept C12590798 @default.
• W2017904568 hasConcept C159047783 @default.
• W2017904568 hasConcept C185592680 @default.

• next