Complexed crystal structure of replication restart primosome protein PriB reveals a novel single-stranded DNA-binding mode

PriB is a primosomal protein required for replication restart in Escherichia coli. PriB stimulates PriA helicase activity via interaction with single-stranded DNA (ssDNA), but the molecular details of this interaction remain unclear. Here, we report the crystal structure of PriB complexed with a 15 bases oligonucleotide (dT15) at 2.7 Å resolution. PriB shares structural similarity with the E.coli ssDNA-binding protein (EcoSSB). However, the structure of the PriB–dT15 complex reveals that PriB binds ssDNA differently. Results from filter-binding assays show that PriB–ssDNA interaction is salt-sensitive and cooperative. Mutational analysis suggests that the loop L₄₅ plays an important role in ssDNA binding. Based on the crystal structure and biochemical analyses, we propose a cooperative mechanism for the binding of PriB to ssDNA and a model for the assembly of the PriA–PriB–ssDNA complex. This report presents the first structure of a replication restart primosomal protein complexed with DNA, and a novel model that explains the interactions between a dimeric oligonucleotide-binding-fold protein and ssDNA.     

Identification of a novel protein, PriB, in Klebsiella pneumoniae

PriB is a primosomal protein required for the reinitiation of replication in bacteria. Here, we report the identification and characterization of a novel PriB protein in Klebsiella pneumoniae (KPN_04595; KpPriB). Unlike the well-studied Escherichia coli PriB protein (EcPriB), which exists as a homodimer comprising 104-aa polypeptides, KpPriB forms a monomer of only 55 aa, due to the absence of the 49 aa N-terminus in KpPriB. Although this N-terminal region (1–49 aa) in EcPriB contains several important residues, such as K18, R34, and W47, which are crucial for ssDNA binding, we found that KpPriB binds ssDNA, but not ssRNA, with comparable affinity as that for EcPriB. Results from filter-binding assays demonstrate that the KpPriB–ssDNA interaction is cooperative and salt-sensitive. Substituting the residue K33 in KpPriB with alanine, the position corresponding to the classic ssDNA-binding residue K82 of EcPriB located in loop L₄₅, significantly reduced ssDNA-binding activity and cooperativity. These results reveal that the 1–49 aa region of the classical PriB protein is unnecessary for ssDNA binding. On the basis of these findings, the structure–function relationships of KpPriB are discussed.     

Interactions of the Escherichia coli Primosomal PriB Protein with the Single-stranded DNA. Stoichiometries, Intrinsic Affinities, Cooperativities, and Base Specificities

Quantitative analysis of the interactions of the Escherichia coli primosomal PriB protein with a single-stranded DNA was done using quantitative fluorescence titration, photocrosslinking, and analytical ultracentrifugation techniques. Stoichiometry studies were done with a series of etheno-derivatives of single-stranded (ss) DNA oligomers. Interactions with the unmodified nucleic acids were studied, using the macromolecular competition titration (MCT) method. The total site-size of the PriB dimer-ssDNA complex, i.e. the maximum number of nucleotides occluded by the PriB dimer in the complex, is 12 ± 1 nt. The protein has a single DNA-binding site, which is located centrally within the dimer and has a functionally homogeneous structure. The stoichiometry and photocrosslinking data show that only a single monomer of the PriB dimer engages in interactions with the nucleic acid. The analysis of the PriB binding to long oligomers was done using a statistical thermodynamic model that takes into account the overlap of potential binding sites and cooperative interactions. The PriB dimer binds the ssDNA with strong positive cooperativity. Both the intrinsic affinity and cooperative interactions are accompanied by a net ion release, with anions participating in the ion exchange process. The intrinsic binding process is an entropy-driven reaction, suggesting strongly that the DNA association induces a large conformational change in the protein. The PriB protein shows a dramatically strong preference for the homo-pyrimidine oligomers with an intrinsic affinity higher by about three orders of magnitude, as compared to the homo-purine oligomers. The significance of these results for PriB protein activity is discussed.     

Insight into the interaction between PriB and DnaT on bacterial DNA replication restart: Significance of the residues on PriB dimer interface and highly acidic region on DnaT

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB−ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66−Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria.     

Diffusion of Human Replication Protein A along Single-Stranded DNA

Replication protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding protein that plays critical roles in most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D₁ ~ 5000 nt² s^− 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA.     

A Single Residue Determines the Cooperative Binding Property of a Primosomal DNA Replication Protein,PriB, to Single-Stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)₂₅, and (StPriB)₂₅ are proposed.     

Ultrafast Redistribution of E. coli SSB along Long Single-Stranded DNA via Intersegment Transfer

Single-stranded DNA binding proteins (SSBs) selectively bind single-stranded DNA (ssDNA) and facilitate recruitment of additional proteins and enzymes to their sites of action on DNA. SSB can also locally diffuse on ssDNA, which allows it to quickly reposition itself while remaining bound to ssDNA. In this work, we used a hybrid instrument that combines single-molecule fluorescence and force spectroscopy to directly visualize the movement of Escherichia coli SSB on long polymeric ssDNA. Long ssDNA was synthesized without secondary structure that can hinder quantitative analysis of SSB movement. The apparent diffusion coefficient of E. coli SSB thus determined ranged from 70,000 to 170,000 nt²/s, which is at least 600 times higher than that determined from SSB diffusion on short ssDNA oligomers, and is within the range of values reported for protein diffusion on double-stranded DNA. Our work suggests that SSB can also migrate via a long-range intersegment transfer on long ssDNA. The force dependence of SSB movement on ssDNA further supports this interpretation.     

Crystal structure of DnaT84–153-dT10 ssDNA complex reveals a novel single-stranded DNA binding mode

DnaT is a primosomal protein that is required for the stalled replication fork restart in Escherichia coli. As an adapter, DnaT mediates the PriA-PriB-ssDNA ternary complex and the DnaB/C complex. However, the fundamental function of DnaT during PriA-dependent primosome assembly is still a black box. Here, we report the 2.83 Å DnaT^84–153-dT10 ssDNA complex structure, which reveals a novel three-helix bundle single-stranded DNA binding mode. Based on binding assays and negative-staining electron microscopy results, we found that DnaT can bind to phiX 174 ssDNA to form nucleoprotein filaments for the first time, which indicates that DnaT might function as a scaffold protein during the PriA-dependent primosome assembly. In combination with biochemical analysis, we propose a cooperative mechanism for the binding of DnaT to ssDNA and a possible model for the assembly of PriA-PriB-ssDNA-DnaT complex that sheds light on the function of DnaT during the primosome assembly and stalled replication fork restart. This report presents the first structure of the DnaT C-terminal complex with ssDNA and a novel model that explains the interactions between the three-helix bundle and ssDNA.     

An electrochemical sensor based on single-stranded DNA-poly(sulfosalicylic acid) composite film for simultaneous determination of adenine, guanine, and thymine

Poly(sulfosalicylic acid) and single-stranded DNA composite (PSSA–ssDNA)-modified glassy carbon electrode (GCE) was prepared by electropolymerization and then successfully used to simultaneously determine adenine (A), guanine (G), and thymine (T). The characterization of electrochemically synthesized PSSA–ssDNA film was investigated by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). The modified electrode exhibited enhanced electrocatalytic behavior and good stability for the simultaneous determination of A, G, and T in 0.1 M phosphate buffer solution (PBS, pH 7.0). Well-separated voltammetric peaks were obtained among A, G, and T presented in the analyte mixture. Under the optimal conditions, the peak currents for A, G, and T increased linearly with the increase of analyte mixture concentration in the ranges of 6.5 × 10⁻⁸ to 1.1 × 10⁻⁶, 6.5 × 10⁻⁸ to 1.1 × 10⁻⁶, and 4.1 × 10⁻⁶ to 2.7 × 10⁻⁵ M, respectively. The detection limits (signal/noise = 3) for A, G, and T were 2.2 × 10^–8, 2.2 × 10^–8, and 1.4 × 10^–6 M, respectively.     

A three-dimensional FRET analysis to construct an atomic model of the actin-tropomyosin complex on a reconstituted thin filament

Fluorescence resonance energy transfer (FRET) was used to construct an atomic model of the actin–tropomyosin (Tm) complex on a reconstituted thin filament. We generated five single-cysteine mutants in the 146–174 region of rabbit skeletal muscle α-Tm. An energy donor probe was attached to a single-cysteine Tm residue, while an energy acceptor probe was located in actin Gln41, actin Cys374, or the actin nucleotide binding site. From these donor–acceptor pairs, FRET efficiencies were determined with and without Ca²⁺. Using the atomic coordinates for F-actin and Tm, we searched all possible arrangements for Tm segment 146–174 on F-actin to calculate the FRET efficiency for each donor–acceptor pair in each arrangement. By minimizing the squared sum of deviations for the calculated FRET efficiencies from the observed FRET efficiencies, we determined the location of the Tm segment on the F-actin filament. Furthermore, we generated a set of five single-cysteine mutants in each of the four Tm regions 41–69, 83–111, 216–244, and 252–279. Using the same procedures, we determined each segment's location on the F-actin filament. In the best-fit model, Tm runs along actin residues 217–236, which were reported to compose the Tm binding site. Electrostatic, hydrogen-bonding, and hydrophobic interactions are involved in actin and Tm binding. The C-terminal region of Tm was observed to contact actin more closely than did the N-terminal region. Tm contacts more residues on actin without Ca²⁺ than with it. Ca²⁺-induced changes on the actin–Tm contact surface strongly affect the F-actin structure, which is important for muscle regulation.     

Characterization of a Single-Stranded DNA-Binding Protein from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as in DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Pseudomonas aeruginosa PAO1 SSB (PaSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified PaSSB by gel filtration chromatography revealed a stable tetramer in solution. In fluorescence titrations, PaSSB bound 22–32 nucleotides (nt) per tetramer depending on salt concentration. Using EMSA, we characterized the stoichiometry of PaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 29 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of PaSSB for the first tetramer were less than those for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding mode of PaSSB is expected to be noncooperative.     

Integrative Modelling Coupled with Ion Mobility Mass Spectrometry Reveals Structural Features of the Clamp Loader in Complex with Single-Stranded DNA Binding Protein

DNA polymerase III, a decameric 420-kDa assembly, simultaneously replicates both strands of the chromosome in Escherichia coli. A subassembly of this holoenzyme, the seven-subunit clamp loader complex, is responsible for loading the sliding clamp (β₂) onto DNA. Here, we use structural information derived from ion mobility mass spectrometry (IM-MS) to build three-dimensional models of one form of the full clamp loader complex, γ₃δδ′ψχ (254 kDa). By probing the interaction between the clamp loader and a single-stranded DNA (ssDNA) binding protein (SSB₄) and by identifying two distinct conformational states, with and without ssDNA, we assemble models of ψχ–SSB₄ (108 kDa) and the clamp loader–SSB₄ (340 kDa) consistent with IM data. A significant increase in measured collision cross-section (~ 10%) of the clamp loader–SSB₄ complex upon DNA binding suggests large conformational rearrangements. This DNA bound conformation represents the active state and, along with the presence of ψχ, stabilises the clamp loader–SSB₄ complex. Overall, this study of a large heteromeric complex analysed by IM-MS, coupled with integrative modelling, highlights the potential of such an approach to reveal structural features of previously unknown complexes of high biological importance.     

Characterization of the Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg²⁺, while ssDNA AP site cleavage required Mg²⁺ (optimally at 0.5-2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3′-PO₄²⁻ group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.     

Characterization of a Single-Stranded DNA Binding Protein from <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Typhimurium LT2

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism, such as DNA replication, repair, and recombination, and is essential for cell survival. We characterized the single-stranded DNA (ssDNA)-binding properties of Salmonella enterica serovar Typhimurium LT2 SSB (StSSB) by using fluorescence quenching measurements and electrophoretic mobility shift analysis (EMSA). Analysis of purified StSSB by gel filtration chromatography showed a stable tetramer in solution. In fluorescence titrations, StSSB bound to 21–38 nucleotides (nt) per tetramer depending on the salt concentration. Using EMSA, we characterized the stoichiometry of StSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined to be 22 ± 1 nt. Furthermore, EMSA results indicated that the dissociation constants of StSSB for the first tetramer were less than that for the second tetramer. On the basis of these biophysical analyses, the ssDNA binding-mode of StSSB is expected to be noncooperative.     

Crystal structure of PriB, a primosomal DNA replication protein of Escherichia coli

PriB is one of the Escherichia coli varphiX-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 A 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 varphiX-type primosome assembly.     

Rat polymerase beta gapped DNA interactions: antagonistic effects of the 5' terminal PO4 - group and magnesium on the enzyme binding to the gapped DNAs with different ssDNA gaps

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO ₄ ⁻ group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg⁺² differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO ₄ ⁻ group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO ₄ ⁻ group increases the stability of internal intermediates with the increasing length of the gap, Mg⁺² decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg⁺² effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme. This work was supported by NIH Grant GM-58565 (to W. B.)     

A single residue determines the cooperative binding property of a primosomal DNA replication protein, PriB, to single-stranded DNA

PriB is a primosomal protein required for re-initiation of replication in bacteria. We characterized and compared the DNA-binding properties of PriB from Salmonella enterica serovar Typhimurium LT2 (StPriB) and Escherichia coli (EcPriB). Only one residue of EcPriB, V6, was different in StPriB (replaced by A6). Previous structural information revealed that this residue is located on the putative dimer-dimer interface of PriB and is not involved in single-stranded DNA (ssDNA) binding. The cooperative binding mechanism of StPriB to DNA is, however, very different from that of EcPriB. Unlike EcPriB, which forms a single complex with ssDNAs of various lengths, StPriB forms two or more distinct complexes. Based on these results, as well as information on structure, binding modes for forming a stable complex of PriB with ssDNA of 25 nucleotides (nt), (EcPriB)25, and (StPriB)25 are proposed.     

Mapping DNA conformations and interactions within the binding cleft of bacteriophage T4 single-stranded DNA binding protein (gp32) at single nucleotide resolution

In this study, we use single-stranded DNA (oligo-dT) lattices that have been position-specifically labeled with monomer or dimer 2-aminopurine (2-AP) probes to map the local interactions of the DNA bases with the nucleic acid binding cleft of gp32, the single-stranded binding (ssb) protein of bacteriophage T4. Three complementary spectroscopic approaches are used to characterize these local interactions of the probes with nearby nucleotide bases and amino acid residues at varying levels of effective protein binding cooperativity, as manipulated by changing lattice length. These include: (i) examining local quenching and enhancing effects on the fluorescence spectra of monomer 2-AP probes at each position within the cleft; (ii) using acrylamide as a dynamic-quenching additive to measure solvent access to monomer 2-AP probes at each ssDNA position; and (iii) employing circular dichroism spectra to characterize changes in exciton coupling within 2-AP dimer probes at specific ssDNA positions within the protein cleft. The results are interpreted in part by what we know about the topology of the binding cleft from crystallographic studies of the DNA binding domain of gp32 and provide additional insights into how gp32 can manipulate the ssDNA chain at various steps of DNA replication and other processes of genome expression.