Absence Of Nucleotide Cofactor Research Articles

Single-molecule characterization of compressed RecA nucleoprotein filaments

RecA is a central enzyme of homologous recombination in bacteria, which plays a major role in DNA repair, natural transformation and SOS-response activation. RecA forms nucleoprotein filaments on single-stranded DNA with a highly conserved architecture that is also shared by eukaryotic recombinases. One of the key features of these filaments is the ability to switch between stretched and compressed conformations in response to ATP binding and hydrolysis. However, the functional role of such conformational changes is not fully understood. Structural data revealed that in the absence of ATP RecA binds DNA with the stoichiometry of 5 nucleotides per one monomer, while in the presence of ATP the binding stoichiometry is 3:1. Such differences suggest incompatibility of the active and inactive conformations, yet dynamic single-molecule studies demonstrated that ATP and apo conformations can be directly interconvertible. In the present work we use a single-molecule approach to address the features of inactive RecA nucleoprotein filaments formed de novo in the absence of nucleotide cofactors. We show that compressed RecA-DNA filaments can exist with both 5:1 and 3:1 binding stoichiometry which is determined by conditions of the filament assembly. However, only a 3:1 stoichiometry allows direct interconvertibility with the active ATP-bound conformation.

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.

Protein–DNA interactions in high speed AFM: single molecule diffusion analysis of human RAD54

High-speed AFM (atomic force microscopy also called scanning force microscopy) provides nanometre spatial resolution and sub-second temporal resolution images of individual molecules. We exploit these features to study diffusion and motor activity of the RAD54 DNA repair factor. Human RAD54 functions at critical steps in recombinational-DNA repair. It is a member of the Swi2/Snf2 family of chromatin remodelers that translocate on DNA using ATP hydrolysis. A detailed single molecular description of DNA-protein interactions shows intermediate states and distribution of variable states, usually hidden by ensemble averaging. We measured the motion of individual proteins using single-particle tracking and observed that random walks were affected by imaging-buffer composition. Non-Brownian diffusion events were characterized in the presence and in the absence of nucleotide cofactors. Double-stranded DNA immobilized on the surface functioned as a trap reducing Brownian motion. Distinct short range slides and hops on DNA were visualized by high-speed AFM. These short-range interactions were usually inaccessible by other methods based on optical resolution. RAD54 monomers displayed a diffusive behavior unrelated to the motor activity.

The Escherichia coli ClpA Molecular Chaperone Self-Assembles into Tetramers

The Escherichia coli ATP-dependent protease, ClpAP, is composed of the hexameric ATPase/protein-unfoldase, ClpA, and the tetradecameric proteolytic component, ClpP. ClpP proteolytically degrades folded proteins only when associated with the motor protein ClpA or ClpX, both of which use ATP binding and/or hydrolysis to unfold and translocate proteins into the tetradecameric serine protease ClpP. In addition to ClpA's role in regulating the proteolytic activity of ClpP, ClpA catalyzes protein unfolding of proteins that display target sequences to "remodel" them, in vivo, for regulatory roles beyond proteolytic degradation. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA first requires nucleoside triphosphate binding to assemble into an oligomeric form with protein substrate binding activity. In addition to this nucleotide driven assembly activity, ClpA self-associates in the absence of nucleoside triphosphate binding. An examination of the energetics of the nucleotide driven assembly process cannot be performed without a thermodynamic model of the self-assembly process in the absence of nucleotide cofactor. Here we report an examination of the self-association properties of the E. coli ClpA protein unfoldase through the application of analytical ultracentrifugation and light scattering techniques, including sedimentation velocity, sedimentation equilibrium, and dynamic light scattering approaches. In contrast to published results, application of these approaches reveals that ClpA exists in a monomer-tetramer equilibrium (300 mM NaCl, 10 mM MgCl(2), and 25 mM HEPES, pH 7.5 at 25 degrees C). The implications of these results for the E. coli ClpA self-association and ligand linked association activities are discussed.

Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.

Papillomavirus E1 helicase assembly maintains an asymmetric state in the absence of DNA and nucleotide cofactors.

Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave's propagation within the AAA+ domains is not necessarily coupled with a strictly sequential hydrolysis of ATP. Since a single ATP hydrolysis event would affect the whole hexamer, such events may simply serve to rectify the direction of the wave's motion.

Calorimetric Analysis of Binding of two Consecutive DNA Strands to RecA Protein Illuminates Mechanism for Recognition Of Homology

RecA protein recognises two complementary DNA strands for homologous recombination. To gain insight into the molecular mechanism, the thermodynamic parameters of the DNA binding have been characterised by isothermal calorimetry. Specifically, conformational changes of protein and DNA were searched for by measuring variations in enthalpy change (ΔH) with temperature (heat capacity change, ΔCp). In the presence of the ATP analogue ATPγS, the ΔH for the binding of the first DNA strand depends upon temperature (large ΔCp) and the type of buffer, in a way that is consistent with the organisation of disordered parts and the protonation of RecA upon complex formation. In contrast, the binding of the second DNA strand occurs without any pronounced ΔCp, indicating the absence of further reorganisation of the RecA–DNA filament. In agreement with these findings, a significant change in the CD spectrum of RecA was observed only upon the binding of the first DNA strand. In the absence of nucleotide cofactor, the ΔH of DNA binding is almost independent of temperature, indicating a requirement for ATP in the reorganisation of RecA. When the second DNA strand is complementary to the first, the ΔH is larger than that for non-complementary DNA strand, but less than the ΔH of the annealing of the complementary DNA without RecA. This small ΔH could reflect a weak binding that may facilitate the dissociation of only partly complementary DNA and thus speed the search for complementary DNA. The ΔH of binding DNA sequences displaying strong base-base stacking is small for both the first and second binding DNA strand, suggesting that the second is also stretched upon interaction with RecA. These results support the proposal that the RecA protein restructures DNA, preparing it for the recognition of a complementary second DNA strand, and that the recognition is due mainly to direct base-base contacts between DNA strands.

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Crystal structure of reverse gyrase: insights into the positive supercoiling of DNA.

Reverse gyrase is the only topoisomerase known to positively supercoil DNA. The protein appears to be unique to hyperthermophiles, where its activity is believed to protect the genome from denaturation. The 120 kDa enzyme is the only member of the type I topoisomerase family that requires ATP, which is bound and hydrolysed by a helicase-like domain. We have determined the crystal structure of reverse gyrase from Archaeoglobus fulgidus in the presence and absence of nucleotide cofactor. The structure provides the first view of an intact supercoiling enzyme, explains mechanistic differences from other type I topoisomerases and suggests a model for how the two domains of the protein cooperate to positively supercoil DNA. Coordinates have been deposited in the Protein Data Bank under accession codes 1GKU and 1GL9.

Archaeal RadA protein binds DNA as both helical filaments and octameric rings

The Escherichia coli RecA protein has been a model for understanding homologous eukaryotic recombination proteins such as Rad51. The active form of both RecA and Rad51 appear to be helical filaments polymerized on DNA, in which an unusual helical structure is induced in the DNA. Surprisingly, the human meiosis-specific homolog of RecA, Dmc1, has thus far only been observed to bind DNA as an octameric ring. Sequence analysis and biochemical studies have shown that archaeal RadA proteins are more closely related to Rad51 and Dmc1 than the bacterial RecA proteins. We find that the Sulfolobus solfataricus RadA protein binds DNA in the absence of nucleotide cofactor as an octameric ring and in the presence of ATP as a helical filament. Since it is likely that RadA is closely related to a common ancestral protein of both Rad51 and Dmc1, the two DNA-binding forms of RadA may provide insight into the divergence that has taken place between Rad51 and Dmc1.

DNA rotation by a coordinated conformational change of RecA filaments.

RecA protein forms two types of filament structures in the presence or absence of nucleotide cofactors. We recently constructed two molecular models of the DNA structure, the unwound form (N-type structure) and the twisted form (S-type structure), which correspond to the helical pitches of the extended form (ATP form) and compressed form (non-nucleotide form) of the RecA filament, respectively. If ATP hydrolysis is unidirectional and cooperative, and if the extended and compressed forms of RecA filament are interconvertible, the coordinated conformational change travels along the filament. While the RecA filament undergoes this worm-like motion, the wave of conformational change between the unwound and the twisted forms of DNA travels as ATP hydrolyzes, which makes the DNA strands rotate.

Purification and characterization of the RecF protein from Bacillus subtilis 168.

Genetic evidence suggests that the Bacillus subtilis recF gene product is involved in DNA repair and recombination. The RecF protein was overproduced and purified. NH2-terminal protein sequence analysis of RecF was consistent with the deduced amino acid sequence of the recF gene. The RecF protein (predicted molecular mass 42.3 kDa) bound single- and double-stranded DNA in a filter binding and in a gel retarding assay. The RecF-ssDNA or -dsDNA complex formation proceeds in the absence of nucleotide cofactors. RecF-ssDNA interaction is markedly stimulated by divalent cations. The apparent equilibrium constants of the RecF-DNA complexes are approximately 110-130 nM for both ssDNA and dsDNA. The binding reaction shows no cooperativity. The RecF protein does not physically interact with the RecR protein. Under our experimental conditions an ATPase activity was not associated with the purified RecF protein or with the RecF and RecR proteins.

Effects of Intercalators on Complexation of RecA with Duplex DNA

To elucidate the binding mode of recombination protein A (RecA) to double-stranded (ds) DNA, the effects on the RecA-DNA interaction of several mono- and bisintercalators of the acridine, phenanthridine, and cyanine classes have been investigated by linear dichroism spectroscopy. Simple monointercalators lacking side chains efficiently promoted the binding of RecA to dsDNA in the absence of nucleotide cofactor, which is otherwise required. Bisintercalators varied in their ability to induce RecA binding, while monointercalators with aminoalkyl side chains proved inefficient. Modification of DNA structure by the intercalator appears to be necessary for induction of RecA binding, but if the intercalator has a bulky minor-groove-binding side chain, it does not induce RecA binding. In detailed studies with acridines, neither the binding geometry of intercalators nor the structure of DNA was significantly modified upon binding of RecA without cofactor. Judged by circular dichroism, similar RecA conformational changes accompanied bis-9-aminoacridine- and ATP gamma S-induced RecA association with DNA. In the presence of ATP gamma S, the intercalators inhibited the rate of RecA binding to dsDNA and were extruded from DNA upon binding of RecA. This competitive aspect may suggest that intercalation of some amino acid residue(s) plays a role in nucleotide-induced RecA binding. The stoichiometry of the RecA-DNA-intercalator filament was determined; in the fully formed filament the base pair:intercalator ratio is 2, and the base pair:RecA ratio also 2. This contrasts with a base pair:RecA ratio of 3 in the ATP gamma S-induced filament, although in both cases the DNA experiences 50% extension.

Spectroscopic Demonstration of a Linkage between the Kinetics of NTP Hydrolysis and the Conformational State of the recA-Single-stranded DNA Complex

We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S0.5 = 70 microM) and purine riboside triphosphate (PTP) (S0.5 = 110 microM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S0.5 = 400 microM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S0.5 (PTP) is increased to 130 microM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S0.5 value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S0.5 of 100-120 microM or lower required for stabilization of the strand exchange-active conformation.

The DNA-Binding Site of the Reca Protein. Photochemical Cross-Linking of Tyrl03 to Single-Stranded DNA

To investigate the DNA-binding site in the Escherichia coli RecA protein, RecA was covalently cross-linked to oligodeoxythymidine [p(dT)14] by irradiation with ultraviolet light. We identified the site of cross-linking of the protein when the RecA.p(dT)14 complex was formed in the absence of nucleotide cofactor as well as in the presence of adenosine 5'-[gamma-thio]triphosphate. When RecA.p(dT)14 complex formed without nucleotide cofactor was irradiated with ultraviolet light, a cross-linked peptide was found after digestion with Achromobactor lyticus protease I. Amino acid composition of the peptide was determined. The results indicated that the site of cross-linking was in the region spanning amino acid residues 89-106. Further digestion of the cross-linked fragment with Staphylococcus aureus V8 protease indicated that Tyr103 was the site of cross-linking. When the complex formed with adenosine 5'-[gamma-thio]triphosphate was irradiated with ultraviolet light, two cross-linked sites were detected, which were in the region of residues 89-106 and residues 178-183. These regions are far from the two disordered loops in the crystal structure, which were suggested to be DNA-binding sites by Story et al. [Story, R. M., Weber, T. W. & Steitz, T. A. (1992) Nature 355, 318-325].

The DNA-Binding Site of the Reca Protein. Photochemical Cross-Linking of Tyrl03 to Single-Stranded DNA

To investigate the DNA-binding site in the Escherichia coli RecA protein, RecA was covalently cross-linked to oligodeoxythymidine [p(dT)14] by irradiation with ultraviolet light. We identified the site of cross-linking of the protein when the RecA.p(dT)14 complex was formed in the absence of nucleotide cofactor as well as in the presence of adenosine 5'-[gamma-thio]triphosphate. When RecA.p(dT)14 complex formed without nucleotide cofactor was irradiated with ultraviolet light, a cross-linked peptide was found after digestion with Achromobactor lyticus protease I. Amino acid composition of the peptide was determined. The results indicated that the site of cross-linking was in the region spanning amino acid residues 89-106. Further digestion of the cross-linked fragment with Staphylococcus aureus V8 protease indicated that Tyr103 was the site of cross-linking. When the complex formed with adenosine 5'-[gamma-thio]triphosphate was irradiated with ultraviolet light, two cross-linked sites were detected, which were in the region of residues 89-106 and residues 178-183. These regions are far from the two disordered loops in the crystal structure, which were suggested to be DNA-binding sites by Story et al. [Story, R. M., Weber, T. W. & Steitz, T. A. (1992) Nature 355, 318-325].

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.

Effect of nucleotide cofactor structure on recA protein-promoted DNA pairing. 2. DNA renaturation reaction.

We have examined the effects of the structurally related nucleoside triphosphates, adenosine triphosphate (ATP), purine riboside triphosphate (PTP), inosine triphosphate (ITP), and guanosine triphosphate (GTP), on the recA protein-promoted DNA renaturation reaction (phi X DNA). In the absence of nucleotide cofactor, the recA protein first converts the complementary single strands into unit-length duplex DNA and other relatively small paired DNA species; these initial products are then slowly converted into more complex multipaired network DNA products. ATP and PTP stimulate the conversion of initial product DNA into network DNA, whereas ITP and GTP completely suppress network DNA formation. The formation of network DNA is also inhibited by all four of the corresponding nucleoside diphosphates, ADP, PDP, IDP, and GDP. Those nucleotides which stimulate the formation of network DNA are found to enhance the formation of large recA-ssDNA aggregates, whereas those which inhibit network DNA formation cause the dissociation of these nucleoprotein aggregates. These results not only implicate the nucleoprotein aggregates as intermediates in the formation of network DNA, but also establish the functional equivalency of ITP and GTP with the nucleoside diphosphates. Additional experiments indicate that the net effect of ITP and GTP on the DNA renaturation reaction is dominated by the corresponding nucleoside diphosphates, IDP and GDP, that are generated by the NTP hydrolysis activity of the recA protein.