Investigation of binding between recA protein and single-stranded polynucleotides with the aid of a fluorescent deoxyribonucleic acid derivative

The availability of epsilon DNA, a fluorescent ssDNA derivative, has made it possible to examine quantitatively the interactions between recA protein and single-stranded polynucleotides. Fluorescence titrations of epsilon DNA with recA protein and vice versa establish that each recA protein monomer covers 5.5 epsilon DNA nucleotides and that the dissociation constant of the recA-epsilon DNA complex is 10 nM. Fluorescence titrations of recA protein-epsilon DNA mixtures with poly(dT) establish that each recA protein monomer covers 5.1 poly(dT) nucleotides and that the dissociation constant of the recA-poly(dT) complex is 0.03 nM. Observations on how the addition of ssDNA affects the fluorescence of recA protein-epsilon DNA mixtures establish that the dissociation constant of the recA-ssDNA complex exceeds 20 microM. Stopped-flow kinetics in which excess recA protein binds to epsilon DNA indicate that k2 = 6 X 10(6) M-1 s-1 for the process. A more approximate kinetic technique indicates that recA protein binds to epsilon DNA at least one-tenth as fast as to poly(dT); the rate constant for dissociation of recA-epsilon DNA exceeds that for recA-poly(dT) by at least 30-fold. epsilon DNA is proven to be a versatile reagent for studying single-stranded polynucleotide-protein interactions. Not only can its own complexes with protein be investigated but also, under suitable circumstances, it can be used as a fluorescent probe to explore complexes incorporating nonfluorescent polynucleotides.     

Purification and characterization of the coliphage N4-coded single-stranded DNA binding protein

We have purified and characterized a single-stranded DNA binding protein (N4 SSB) induced after coliphage N4 infection. It has a monomeric molecular weight of 31,000 and contains 10 tyrosine and 1-2 tryptophan amino acid residues. Its fluorescence spectrum is dominated by the tyrosine residues, and their fluorescence is quenched when the protein binds single-stranded DNA. Fluorescence quenching was used as an assay to quantitate binding of the protein to single-stranded nucleotides. The N4 single-stranded DNA binding protein binds cooperatively to single-stranded nucleic acids and binds single-stranded DNA more tightly than RNA. The binding involves displacement of cations from the DNA and anions from the protein. The apparent binding affinity is very salt-dependent, decreasing as much as 1,000-fold for a 10-fold increase in NaCl concentration. The degree of cooperativity (omega) is relatively independent of salt concentration. At 37 degrees C in 0.22 M NaCl, the protein has an intrinsic binding constant for M13 viral DNA of 3.8 x 10(4) M-1, a cooperativity factor omega of 300, and binding site size of 11 nucleotides per monomer. The protein lowers the melting point of poly(dA.dT).poly(dA-dT) by greater than 60 degrees C but cannot lower the melting transition or assist in the renaturation of natural DNA. N4 single-stranded DNA binding protein enhances the rate of DNA synthesis catalyzed by the N4 DNA polymerase by increasing the processivity of the N4 DNA polymerase and melting out hairpin structures that block polymerization.     

Interactions of the KWK6 cationic peptide with short nucleic acid oligomers: demonstration of large Coulombic end effects on binding at 0.1-0.2 M salt

We quantify Coulombic end effects (CEE) on oligocation-nucleic acid interactions at salt concentrations ([salt]) in the physiological range. Binding constants (K(obs); per site, at zero binding density) for the +8-charged C-amidated oligopeptide KWK6 and short single-stranded DNA oligonucleotides [dTpdT(|Z(D)|), where 6 < or = |Z(D)| < or = 22 is the number of DNA phosphates] were determined as a function of [salt] by fluorescence quenching. For the different DNA oligomers, K(obs) values are similar at high [salt], but diverge as [salt] decreases because -S(a)K(obs) identical with--partial partial differential ln K(obs)/ partial differential ln a+/- increases strongly with |Z(D)|. For binding of KWK6 near 0.1 M salt, -S(a)K(obs) is 5.5 +/- 0.2 for dT(pdT)22, 4.0 +/- 0.2 for dT(pdT)10 and 2.9 +/- 0.2 for dT(pdT)6, as compared with 6.5 +/- 0.3 for poly(dT). Similarly, at 0.1 M salt, K(obs) per site for poly(dT) exceeds K(obs) for dT(pdT)22 by 7-fold, for dT(pdT)10 by 50-fold and for dT(pdT)6 by 700-fold. We interpret the reductions in K(obs) and |S(a)K(obs)| with decreasing |Z(D)| as a significant CEE that causes binding to the terminal regions of a nucleic acid to be weaker and less salt dependent than interior binding. We analyze long oligonucleotide-KWK6 binding data in terms of a trapezoidal model for the local (axial) salt cation concentration on single-stranded DNA to estimate the size of the CEE to be at least seven phosphates on each end at 0.1 M salt.     

<Emphasis Type="Italic">Escherichia coli</Emphasis> AlkB interacts with single-stranded DNA binding protein SSB by an intrinsically disordered region of SSB

Intrinsically disordered regions (IDRs) of proteins often regulate function through interactions with folded domains. Escherichia coli single-stranded DNA binding protein SSB binds and stabilizes single-stranded DNA (ssDNA). The N-terminal of SSB contains characteristic OB (oligonucleotide/oligosaccharide-binding) fold which binds ssDNA tightly but non-specifically. SSB also forms complexes with a large number proteins via the C-terminal interaction domain consisting mostly of acidic amino acid residues. The amino acid residues located between the OB-fold and C-terminal acidic domain are known to constitute an IDR and no functional significance has been attributed to this region. Although SSB is known to bind many DNA repair protein, it is not known whether it binds to DNA dealkylation repair protein AlkB. Here, we characterize AlkB SSB interaction and demonstrate that SSB binds to AlkB via the IDR. We have established that AlkB-SSB interaction by in vitro pull-down and yeast two-hybrid analysis. We mapped the site of contact to be the residues 152–169 of SSB. Unlike most of the SSB-binding proteins which utilize C-terminal acidic domain for interaction, IDR of SSB is necessary and sufficient for AlkB interaction.     

recA protein filaments bind two molecules of single-stranded DNA with off rates regulated by nucleotide cofactor

To probe the role of nucleotide cofactor in the binding of single-stranded DNA to recA protein, we have developed a sedimentation assay using 5'-labeled 32P-poly(dT).recA.poly(dT) complexes sediment quantitatively when centrifuged at 100,000 x g for 45 min, whereas free poly(dT) remains in the supernatant. In the presence of ATP, between 6 and 7 bases cosediment per recA monomer; but when ADP is present or in the absence of added nucleotide cofactor, only 3-3.5 bases/recA monomer cosediment. In competition experiments in which recA.32P-poly(dT) complexes are incubated with unlabeled poly(dT), we again find 3-3.5 bases of labeled poly(dT) cosedimenting per recA monomer when no nucleotide cofactor is present. However, when the same experiment is performed with ATP, only half of the expected 6-7 bases of labeled poly(dT) remain bound to the DNA, demonstrating that half of the poly(dT) in the complex exchanges rapidly with free poly(dT), whereas the other half equilibrates slowly, like poly(dT) in the absence of nucleotide. The rate of exchange of the second more tightly bound poly(dT) is accelerated when ADP is present. Our observations are rationalized by a model in which each recA protein helical filament binds two strands of poly(dT) with a stoichiometry of 3-3.5 bases/recA monomer/strand.     

Site-directed mutagenesis reveals the thermodynamic requirements for single-stranded DNA recognition by the telomere-binding protein Cdc13

The essential Saccharomyces cerevisiae protein Cdc13 binds the conserved single-stranded overhang at the end of telomeres and mediates access of protein complexes involved in both end-capping and telomerase activity. The single-stranded DNA-binding domain (ssDBD) of Cdc13 exhibits both high affinity (K(d) of 3 pM) and sequence specificity for the GT-rich sequences present at yeast telomeres. We have used the ssDBD of Cdc13 to understand the sequence-specific recognition of extended single-stranded DNA (ssDNA). The recent structure of the Cdc13 DNA-binding domain revealed that ssDNA is recognized by a large protein surface containing an oligonucleotide/oligosaccharide-binding fold (OB-fold) augmented by an extended 30-amino acid loop. Contacts to ssDNA occur via a contiguous surface of aromatic, hydrophobic, and basic residues. A complete alanine scan of the binding interface has been used to determine the contribution of each contacting side chain to binding affinity. Substitution of any aromatic or hydrophobic residue at the interface was deleterious to binding (20 to >700-fold decrease in binding affinity), while tolerance for replacement of basic residues was observed. The important aromatic and hydrophobic contacts are spread throughout the extended interface, indicating that the entire surface is both structurally and thermodynamically required for binding. While all of these contacts are important, several of the individual alanine substitutions that abolish binding cluster to one region of the protein surface. This region is vital for recognition of four bases at the 5' end of the DNA and constitutes a "hotspot" of binding affinity.     

Replication protein A interactions with DNA. 1. Functions of the DNA-binding and zinc-finger domains of the 70-kDa subunit

Human replication protein A (RPA) is a multiple subunit single-stranded DNA-binding protein that is required for multiple processes in cellular DNA metabolism. This complex, composed of subunits of 70, 32, and 14 kDa, binds to single-stranded DNA (ssDNA) with high affinity and participates in multiple protein-protein interactions. The 70-kDa subunit of RPA is known to be composed of multiple domains: an N-terminal domain that participates in protein interactions, a central DNA-binding domain (composed of two copies of a ssDNA-binding motif), a putative (C-X2-C-X13-C-X2-C) zinc finger, and a C-terminal intersubunit interaction domain. A series of mutant forms of RPA were used to elucidate the roles of these domains in RPA function. The central DNA-binding domain was necessary and sufficient for interactions with ssDNA; however, adjacent sequences, including the zinc-finger domain and part of the N-terminal domain, were needed for optimal ssDNA-binding activity. The role of aromatic residues in RPA-DNA interactions was examined. Mutation of any one of the four aromatic residues shown to interact with ssDNA had minimal effects on RPA activity, indicating that individually these residues are not critical for RPA activity. Mutation of the zinc-finger domain altered the structure of the RPA complex, reduced ssDNA-binding activity, and eliminated activity in DNA replication.     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase.

The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity. The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif. Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved. Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC). Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure. The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking. Magnesium is required for primase-ssDNA binding. The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases. The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding. Some possible sources of this gain in entropy are discussed.     

Escherichia coli thioredoxin stabilizes complexes of bacteriophage T7 DNA polymerase and primed templates

The DNA polymerase activity induced after bacteriophage T7 infection of Escherichia coli is found in a complex of two proteins, the T7 gene 5 protein and a host protein, thioredoxin. Gene 5 protein is a DNA polymerase and a 3' to 5' exonuclease. Thioredoxin binds tightly to the gene 5 protein and increases the processivity of polymerization some 1000-fold. Gene 5 protein forms a short-lived complex with the primer-template, poly(dA).oligo(dT), in the absence of Mg2+ and nucleotides. Thioredoxin increases the half-life of the preformed primer-template-polymerase complex from less than a second to approximately 5 min. The dissociation is accelerated by excess single-stranded DNA in an apparent second order reaction, indicating direct transfer of polymerase between DNA fragments. Thioredoxin also reduces the equilibrium dissociation constant, Kd, of the gene 5 protein -poly(dA).oligo(dT) complex 20- to 80-fold. The salt dependence of Kd indicates that thioredoxin stabilizes the primer-template-polymerase complex mainly through additional charge-charge interactions, increasing the estimated number of interactions from 2 to 7. The affinity of gene 5 protein for single-stranded DNA is at least 1000-fold higher than for double-stranded DNA and is little affected by thioredoxin. Under conditions of steady state synthesis the effect of thioredoxin on the polymerization rate is determined by two competing factors, an increase in processivity and a decrease of the dissociation rate of polymerase and replicated template.     

Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors

We have investigated quantitative molecular aspects of the interaction of recA protein with single-stranded DNA, by using a fluorescent modified-DNA referred to as etheno-M13 DNA. In addition, the effects of the nucleotide cofactors ATP and ADP, and the analogues ATP-gamma-S, AMP-P-C-P, and AMP-P-N-P on this interaction have been studied. It is shown that ATP, AMP-P-N-P and, in particular, ATP-gamma-S significantly increase the affinity of recA protein for single-stranded DNA, whereas ADP and, to a lesser degree, AMP-P-C-P decrease the affinity. Binding to etheno-M13 single-stranded DNA is co-operative, with the value of the co-operativity parameter, omega, being approximately 50 under all conditions measured. The effect that ADP has on recA protein-DNA affinity is to lower the intrinsic binding constant, but it has no effect on the co-operativity of binding. In addition, the stability of the recA protein-DNA complex is very salt dependent (d log K/d log [NaC1] approximately -10) and it is the intrinsic binding affinity rather than the co-operativity of binding that is affected; thus, under all conditions observed, recA protein binds single-stranded DNA co-operatively with a value of omega = 50 +/- 10. The binding affinity is also influenced by the type of anion present, being approximately 10,000-fold higher when acetate ion is present instead of chloride ion. These data have been interpreted to suggest that recA protein forms up to five ionic interactions when it binds to single-stranded DNA and that five to six anions are displaced upon binding. The modulation of recA protein-DNA complex stability by nucleotide cofactors suggests that these cofactors play a role in the cycling of recA protein on and off single-stranded DNA, with ATP being required for DNA binding under physiological conditions and ADP serving as a "release" factor. These results are discussed in terms of a model for the role of ATP hydrolysis in a recA protein-single stranded DNA binding cycle.     

Amino acid 55 plays a central role in tetramerization and function of Escherichia coli single-stranded DNA binding protein

The histidine at position 55 of the amino acid sequence of the Escherichia coli single-stranded DNA binding protein was replaced by tyrosine, glutamic acid, lysine, phenylalanine, and isoleucine. The properties of the mutant proteins were determined using analytical ultracentrifugation, NMR spectroscopy, gel filtration, and fluorimetric detection of their single-stranded DNA binding ability. While the phenylalanine and isoleucine substitutions did not change the properties of the protein measurably, tyrosine and lysine mutants dissociate into subunits and loose some of their binding affinity for poly(dT). For the lysine mutant we show by electron microscopy that the protein, although fully dissociated and possibly denatured in the free state, binds to poly(dT) as a tetramer indistinguishable from the wild-type protein. The process of tetramerization as observed via single-stranded DNA binding ability is composed of a variety of steps ranging in time from some milliseconds to several hours; it probably involves several forms of dissociated and non-native protein.     

Roles of active site residues and the HUH motif of the F plasmid TraI relaxase

Bacterial conjugation, transfer of a single strand of a conjugative plasmid between bacteria, requires sequence-specific single-stranded DNA endonucleases called relaxases or nickases. Relaxases contain an HUH (His-hydrophobe-His) motif, part of a three-His cluster that binds a divalent cation required for the cleavage reaction. Crystal structures of the F plasmid TraI relaxase domain, with and without bound single-stranded DNA, revealed an extensive network of interactions involving HUH and other residues. Here we study the roles of these residues in TraI function. Whereas substitutions for the three His residues alter metal-binding properties of the protein, the same substitution at each position elicits different effects, indicating that the residues contribute asymmetrically to metal binding. Substitutions for a conserved Asp that interacts with one HUH His demonstrate that the Asp modulates metal affinity despite its distance from the metal. The bound metal enhances binding of ssDNA to the protein, consistent with a role for the metal in positioning the scissile phosphate for cleavage. Most substitutions tested caused significantly reduced in vitro cleavage activities and in vivo transfer efficiencies. In summary, the results suggest that the metal-binding His cluster in TraI is a finely tuned structure that achieves a sufficient affinity for metal while avoiding the unfavorable electrostatics that would result from placing an acidic residue near the scissile phosphate of the bound ssDNA.     

Vital roles of the second DNA-binding site of Rad52 protein in yeast homologous recombination

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.     

Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B' motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B' as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.     

Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein.

Bacteriophage T7 gene 2.5 protein has been purified to homogeneity from cells overexpressing its gene. Native gene 2.5 protein consists of a dimer of two identical subunits of molecular weight 25,562. Gene 2.5 protein binds specifically to single-stranded DNA with a stoichiometry of approximately 7 nucleotides bound per monomer of gene 2.5 protein; binding appears to be noncooperative. Electron microscopic analysis shows that gene 2.5 protein is able to disrupt the secondary structure of single-stranded DNA. The single-stranded DNA is extended into a chain of gene 2.5 protein dimers bound along the DNA. In fluorescence quenching and nitrocellulose filter binding assays, the binding constants of gene 2.5 protein to single-stranded DNA are 1.2 x 10(6) M-1 and 3.8 x 10(6) M-1, respectively. Escherichia coli single-stranded DNA-binding protein and phage T4 gene 32 protein bind to single-stranded DNA more tightly by a factor of 25. Fluorescence spectroscopy suggests that tyrosine residue(s), but not tryptophan residues, on gene 2.5 protein interacts with single-stranded DNA.