Specificity of binding of single-stranded DNA-binding protein to its target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB-DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB-DNA complexes. We designed hybrid DNA substrates with 5'- and 3'-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that Escherichia coli SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg(2+) cations or a high ionic strength. In the absence of Mg(2+) cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg(2+) cations.     

SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

A model for oligomeric regulation of APOBEC3G cytosine deaminase-dependent restriction of HIV

APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C --> U deaminations on single-stranded DNA (ssDNA) with marked 3' --> 5' deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3'-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3'-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.     

Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.     

Role of magnesium ion in the interaction between chromomycin A3 and DNA: binding of chromomycin A3-Mg2+ complexes with DNA.

Chromomycin A3 is an antitumor antibiotic which blocks macromolecular synthesis via reversible interaction with DNA template only in the presence of divalent metal ions such as Mg2+. The role of Mg2+ in this antibiotic-DNA interaction is not well understood. We approached the problem in two steps via studies on the interaction of (i) chromomycin A3 and Mg2+ and (ii) chromomycin A3-Mg2+ complex(es) and DNA. Spectroscopic techniques such as absorption, fluorescence, and CD were employed for this purpose. The results could be summed up in two parts. Absorption, fluorescence, and CD spectra of the antibiotic change upon addition of Mg2+ due to complex formation between them. Analysis of the quantitative dependence of change in absorbance of chromomycin A3 (at 440 nm) upon input concentration of Mg2+ indicates formation of two types of complexes with different stoichiometries and formation constants. Trends in change of fluorescence and CD spectroscopic features of the antibiotic in the presence of Mg2+ at different concentrations further corroborate this result. The two complexes are referred to as complex I (with 1:1 stoichiometry in terms of chromomycin A3:Mg2+) and complex II (with 2:1 stoichiometry in terms of chromomycin A3:Mg2+), respectively, in future discussions. The interactions of these complexes with calf thymus DNA were examined to check whether they bind differently to the same DNA. Evaluation of binding parameters, intrinsic binding constants, and binding stoichiometry, by means of spectrophotometric and fluorescence titrations, shows that they are different. Distinctive spectroscopic features of complexes I and II, when they are bound to DNA, also support that they bind differently to the above DNA. Measurement of thermodynamic parameters characterizing their interactions with calf thymus DNA shows that complex I-DNA interaction is exothermic, in contrast to complex II-DNA interaction, which is endothermic. This feature implies a difference in the molecular nature of the interactions between the complexes and calf thymus DNA. These observations are novel and significant to understand the antitumor property of the antibiotic. They are also discussed to provide explanations for the earlier reports that in some cases appeared to be contradictory.     

Observation of single-stranded DNA on mica and highly oriented pyrolytic graphite by atomic force microscopy

Atomic force microscopy was used to image single-stranded DNA (ssDNA) adsorbed on mica modified by Mg(2+), by 3-aminopropyltriethoxysilane or on modified highly oriented pyrolytic graphite (HOPG). ssDNA molecules on mica have compact structures with lumps, loops and super twisting, while on modified HOPG graphite ssDNA molecules adopt a conformation without secondary structures. We have shown that the immobilization of ssDNA under standard conditions on modified HOPG eliminates intramolecular base-pairing, thus this method could be important for studying certain processes involving ssDNA in more details.     

MD simulation and experimental evidence for Mg²+ binding at the B site in human AP endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1), a central enzyme in the base excision repair pathway, cleaves damaged DNA in Mg(2+) dependent reaction. Despite characterization of nine X-ray crystallographic structures of human APE1, in some cases, bound to various metal ions and substrate/product, the position of the metal ion and its stoichiometry for the cleavage reaction are still being debated. While a mutation of the active site E96Q was proposed to eliminate Mg(2+) binding at the "A" site, we show experimentally that this mutant still requires Mg(2+) at concentration similar to that for the wild type enzyme to cleave the AP site in DNA. Molecular dynamics simulations of the wild type APE1, E96Q and a double missense mutant E96Q + D210N indicate that Mg(2+) placed at the A-site destabilizes the bound AP site-containing DNA. In these simulations, the H-bond chain D238-H309-AP site oxygen is broken and the substrate DNA is shifted away from its crystal structure position (1DE9). In contrast, simulations with the Mg(2+) at site B or A+B sites leave the substrate DNA at the position shown in the crystal structure (1DE9). Taken together our MD simulations and biochemical analysis suggests that Mg(2+) binding at the B site is involved in the reaction mechanism associated with endonuclease function of APE1.     

Interaction of mithramycin with chromatin

Mithramycin (MTR) is an anti-cancer antibiotic that blocks the macromolecular biosynthesis via reversible interaction with DNA template in the presence of bivalent metal ion such as Mg2+. In absence of DNA, mithramycin forms two types of complexes with Mg2+, complex I (with 1:1 stoichiometry in terms of MTR: Mg2+) and complex II (with 1:2 stoichiometry in terms of MTR: Mg2+). In an eukaryotic system, the drug would interact with chromatin, a protein-DNA complex. We have employed the spectroscopic techniques such as absorption and fluorescence to study the interaction of MTR: Mg2+ complexes with rat liver chromatin. In this report, we have shown that the two types of ligands have different binding potentials with the same chromatin. This supports our proposition that complexes I and II, are different molecular species. We have also shown that the histone protein(s) reduce the binding potential and the number of available sites for both ligands.     

Dynamic structures of Bacillus subtilis RecN-DNA complexes

Genetic and cytological evidences suggest that Bacillus subtilis RecN acts prior to and after end-processing of DNA double-strand ends via homologous recombination, appears to participate in the assembly of a DNA repair centre and interacts with incoming single-stranded (ss) DNA during natural transformation. We have determined the architecture of RecN–ssDNA complexes by atomic force microscopy (AFM). ATP induces changes in the architecture of the RecN–ssDNA complexes and stimulates inter-complex assembly, thereby increasing the local concentration of DNA ends. The large CII and CIII complexes formed are insensitive to SsbA (counterpart of Escherichia coli SSB or eukaryotic RPA protein) addition, but RecA induces dislodging of RecN from the overhangs of duplex DNA molecules. Reciprocally, in the presence of RecN, RecA does not form large RecA–DNA networks. Based on these results, we hypothesize that in the presence of ATP, RecN tethers the 3′-ssDNA ends, and facilitates the access of RecA to the high local concentration of DNA ends. Then, the resulting RecA nucleoprotein filaments, on different ssDNA segments, might promote the simultaneous genome-wide homology search.     

Differential interactions of antitumor antibiotics chromomycin A(3) and mithramycin with d(TATGCATA)(2) in presence of Mg(2+)

The antitumor antibiotics chromomycin A(3) (CHR) and mithramycin (MTR) are known to inhibit macromolecular biosynthesis by reversibly binding to double stranded DNA with a GC base specificity via the minor groove in the presence of a divalent cation such as Mg(2+). Earlier reports from our laboratory showed that the antibiotics form two types of complexes with Mg(2+): complex I with 1:1 stoichiometry and complex II with 2:1 stoichiometry in terms of the antibiotic and Mg(2+). The binding potential of an octanucleotide, d(TATGCATA)(2), which contains one potential site of association with the above complexes of the two antibiotics, was examined using spectroscopic techniques such as absorption, fluorescence, and circular dichroism. We also evaluated thermodynamic parameters for the interaction. In spite of the presence of two structural moieties of the antibiotic in complex II, a major characteristic feature was the association of a single ligand molecule per molecule of octameric duplex in all cases. This indicated that the modes of association for the two types of complexes with the oligomeric DNA were different. The association was dependent on the nature of the antibiotics. Spectroscopic characterization along with analysis of binding and thermodynamic parameters showed that differences in the mode of recognition by complexes I and II of the antibiotics with polymeric DNA existed at the oligomeric level. Analysis of the thermodynamic parameters led us to propose a partial accommodation of the ligand in the groove without the displacement of bound water molecules and supported earlier results on the DNA structural transition from B --> A type geometry as an obligatory requirement for the accommodation of the bulkier complex II of the two drugs. The role of the carbohydrate moieties of the antibiotics in the DNA recognition process was indicated when we compared the DNA binding properties with the same type of Mg(2+) complex for the two antibiotics.     

Biochemical Characterization of Human Tyrosyl-DNA Phosphodiesterase 2 (TDP2/TTRAP): A Mg2+/Mn2+-DEPENDENT PHOSPHODIESTERASE SPECIFIC FOR THE REPAIR OF TOPOISOMERASE CLEAVAGE COMPLEXES

TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)m(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.     

Structure-based analysis of the metal-dependent mechanism of H-N-H endonucleases

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.     

A 2'-methyl or 2'-methylene group at G+1 in precursor tRNA interferes with Mg2+ binding at the enzyme-substrate interface in E-S complexes of E. coli RNase P

We analyzed processing of precursor tRNAs carrying a single 2'-deoxy, 2'-OCH(3), or locked nucleic acid (LNA) modification at G+1 by Escherichia coli RNase P RNA in the absence and presence of its protein cofactor. The extra methyl or methylene group caused a substrate binding defect, which was rescued at higher divalent metal ion (M(2+)) concentrations (more efficiently with Mn(2+) than Mg(2+)), and had a minor effect on cleavage chemistry at saturating M(2+) concentrations. The 2'-OCH(3) and LNA modification at G+1 resulted in higher metal ion cooperativity for substrate binding to RNase P RNA without affecting cleavage site selection. This indicates disruption of an M(2+) binding site in enzyme-substrate complexes, which is compensated for by occupation of alternative M(2+) binding sites of lower affinity. The 2'-deoxy modification at G+1 caused at most a two-fold decrease in the cleavage rate; this mild defect relative to 2'-OCH(3) and LNA at G+1 indicates that the defect caused by the latter two is steric in nature. We propose that the 2'-hydroxyl at G+1 in the substrate is in the immediate vicinity of the M(2+) cluster at the phosphates of A67 to U69 in helix P4 of E. coli RNase P RNA.     

Magnesium-cationic dummy atom molecules enhance representation of DNA polymerase beta in molecular dynamics simulations: improved accuracy in studies of structural features and mutational effects

Human DNA polymerase beta (pol beta) fills gaps in DNA as part of base excision DNA repair. Due to its small size it is a convenient model enzyme for other DNA polymerases. Its active site contains two Mg(2+) ions, of which one binds an incoming dNTP and one catalyzes its condensation with the DNA primer strand. Simulating such binuclear metalloenzymes accurately but computationally efficiently is a challenging task. Here, we present a magnesium-cationic dummy atom approach that can easily be implemented in molecular mechanical force fields such as the ENZYMIX or the AMBER force fields. All properties investigated here, namely, structure and energetics of both Michaelis complexes and transition state (TS) complexes were represented more accurately using the magnesium-cationic dummy atom model than using the traditional one-atom representation for Mg(2+) ions. The improved agreement between calculated free energies of binding of TS models to different pol beta variants and the experimentally determined activation free energies indicates that this model will be useful in studying mutational effects on catalytic efficiency and fidelity of DNA polymerases. The model should also have broad applicability to the modeling of other magnesium-containing proteins.     

Characterization of two highly similar Rad51 homologs of Physcomitrella patens

The moss Physcomitrella patens, which is a land plant with efficient homologous recombination, encodes two Rad51 proteins (PpaRad51.1 and PpaRad51.2). The PpaRad51.1 and PpaRad51.2 proteins, which share 94 % identity between them, interact with themselves and with each other. Both proteins bind ssDNA and dsDNA in a Mg(2+) and pH-dependent manner, with a stoichiometry of one PpaRad51.1 monomer per 3(+/-1) nt or bp and one PpaRad51.2 monomer per 1(+/-0.5) nt or bp, respectively. At neutral pH, a 1.6-fold excess of both proteins is required for ssDNA and dsDNA binding. PpaRad51.1 and PpaRad51.2 show ssDNA-dependent ATPase activity and efficiently promote strand annealing in a nucleotide-independent but in a Mg(2+)-dependent manner. Both proteins promote joint-molecule formation, DNA strand invasion and are able to catalyse strand exchange in the presence of Mg(2+) and ATP. No further increase in the activities is observed when both proteins are present in the same reaction. None of the PpaRad51 gene products complement the DNA repair and recombination phenotype of Saccharomyces cerevisiae rad51delta mutants. However, PpaRad51.1 confers a dominant-negative DNA repair phenotype, and both PpaRad51 proteins reduce the levels of double-strand break-induced recombination when overexpressed in S. cerevisiae wt cells. These results suggest that both PpaRad51 proteins are bona fide Rad51 proteins that may contribute, in a different manner, to homologous recombination, and that they might replace ScRad51 in a hypothetical yeast protein complex inactivating different functions required for recombinational repair.     

SSB蛋白的高效表达及其与ssDNA的相互作用

大肠杆菌单链结合蛋白SSB在DNA复制、重组和修复中起着重要作用。为研究单链结合蛋白SSB的体外生物功能构建了融合蛋白SSB的表达载体并使其高效表达及易于纯化。ssb基因片段是以E.coli K-12基因组为模板经PCR扩增获得,并通过基因的体外拼接成功构建了表达载体pQE30-ssb。重组菌株M15/ pQE30-ssb经过IPTG的诱导表达了蛋白SSB。收集菌体细胞、超声波破碎后离心取上清进行SDS-PAGE分析,结果表明有一与预期分子量（20.6 kD）相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在。利用固定化金属离子（Ni2+）配体亲和层析柱纯化融合蛋白SSB,其纯度达到90％。通过凝胶层析和等离子共振技术对SSB的生物功能进行了系统研究分析。结果表明,SSB蛋白以四聚体形式与单链DNA分子结合,其亲和力常数（KD）为4.79×10-7 M。     

Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg(2+); seven of these are in the prototypical metal-binding motifs A and B (HX(3)HX(n)()E) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn(2+) or Mg(2+), but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn(2+) than that by Mg(2+); catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn(2+) and Mg(2+) are increased, but the effect of Mn(2+) is 2-fold greater than that of Mg(2+) in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn(2+) or Mg(2+) support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX(3)HX(n)()E motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.