Crystal structure of thermostable DNA photolyase: pyrimidine-dimer recognition mechanism.

DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus, the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.

Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain.

A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degrees C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5'-->3' exonuclease activity, which was enhanced by increasing the temperature to 50 degrees C. Mg(2+), Mn(2+) or Co(2+) ions were required to activate both proteins, whereas Ca(2+) and Zn(2+) had no effects.

Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product.

The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.

Structural genomics projects in Japan.

Two major structural genomics projects exist in Japan. The oldest, the RIKEN Structural Genomics Initiative, has two major goals: to determine bacterial, mammalian, and plant protein structures by X-ray crystallography and NMR spectroscopy and to perform functional analyses with the target proteins. The newest, the structural genomics project at the Biological Information Research Center, focuses on human membrane proteins.

Observation of RecA protein monomer by small angle X-ray scattering with synchrotron radiation.

RecA protein is capable of forming homo-oligomers in solution. The oligomeric and monomeric states of Thermus thermophilus RecA protein were studied by small angle X-ray scattering, a direct method used to measure the overall dimensions of a macromolecule. In the presence of 3 M urea or 0.2 M lithium perchlorate, RecA dissociates from higher oligomeric states to form a hexamer with a radius of gyration (R(g)) of 52 A. The value of R(g) decreased to 36 A at a higher lithium perchlorate concentration (1.0 M). The zero angle intensity, I(0), was consistent with the identification of the former state as a hexamer and the latter as a monomer.

Interaction of UvrA and UvrB proteins with a fluorescent single-stranded DNA. Implication for slow conformational change upon interaction of UvrB with DNA.

UvrA and UvrB proteins play key roles in the damage recognition step in the nucleotide excision repair. However, the molecular mechanism of damage recognition by these proteins is still not well understood. In this work we analyzed the interaction between single-stranded DNA (ssDNA) labeled with a fluorophore tetramethylrhodamine (TMR) and Thermus thermophilus HB8 UvrA (ttUvrA) and UvrB (ttUvrB) proteins. TMR-labeled ssDNA (TMR-ssDNA) as well as UV-irradiated ssDNA stimulated ATPase activity of ttUvrB more strongly than did normal ssDNA, indicating that this fluorescent ssDNA was recognized as damaged ssDNA. The addition of ttUvrA or ttUvrB enhanced the fluorescence intensity of TMR-ssDNA, and the intensity was much greater in the presence of ATP. Fluorescence titration indicated that ttUvrA has higher specificity for TMR-ssDNA than for normal ssDNA in the absence of ATP. The ttUvrB showed no specificity for TMR-ssDNA, but it took over 200 min for the fluorescence intensity of the ttUvrB-TMR-ssDNA complex to reach saturation in the presence of ATP. This time-dependent change could be separated into two phases. The first phase was rapid, whereas the second phase was slow and dependent on ATP hydrolysis. Time dependence of ATPase activity and fluorescence polarization suggested that changes other than the binding reaction occurred during the second phase. These results strongly suggest that ttUvrB binds ssDNA quickly and that a conformational change in ttUrvB-ssDNA complex occurs slowly. We also found that DNA containing a fluorophore as a lesion is useful for directly investigating the damage recognition by UvrA and UvrB.

Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues.

The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain.

The RadA protein from a hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent ATPase that exhibits two disparate catalytic modes, with a transition temperature at 75 degrees C.

The radA gene is an archaeal homolog of bacterial recA and eukaryotic RAD51 genes, which are critical components in homologous recombination and recombinational DNA repair. We cloned the radA gene from a hyperthermophilic archaeon, Pyrobaculum islandicum, overproduced the radA gene product in Escherichia coli and purified it to homogeneity. The purified P. islandicum RadA protein maintained its secondary structure and activities in vitro at high temperatures, up to 87 degrees C. It also showed high stability of 18.3 kcal.mol-1 (76.5 kJ.mol-1) at 25 degrees C and neutral pH. P. islandicum RadA exhibited activities typical of the family of RecA-like proteins, such as the ability to bind ssDNA, to hydrolyze ATP in a DNA-dependent manner and to catalyze DNA strand exchange. At 75 degrees C, all DNAs tested stimulated ATPase activity of the RadA. The protein exhibited a break in the Arrhenius plot of ATP hydrolysis at 75 degrees C. The cooperativity of ATP hydrolysis and ssDNA-binding ability of the protein above 75 degrees C were higher than at lower temperatures, and the activation energy of ATP hydrolysis was lower above this break point temperature. These results suggest that the ssDNA-dependent ATPase activity of P. islandicum RadA displays a temperature-dependent capacity to exist in two different catalytic modes, with 75 degrees C being the critical threshold temperature.

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair.

In the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 A resolution. UvrB comprises four domains, two of which have an alpha/beta structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an alpha-helical domain and a domain consisting of antiparallel beta-sheets (beta-domain). The sequence similarity suggests that the beta-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.

Crystallization and preliminary X-ray diffraction studies of a DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8.

A DNA excision repair enzyme, UvrB, from Thermus thermophilus HB8 was crystallized by the vapor-diffusion method using lithium sulfate as the precipitant and beta-octylglucoside as an additive. The crystals belong to the trigonal space group P3121 or P3221, with unit-cell dimensions of a = b = 136.0 and c = 108.1 A. The crystal is most likely to contain one UvrB protein in an asymmetric unit with the Vm value of 3.8 A3 Da-1. The crystals diffracted X-rays beyond 2.9 A resolution. Although the crystals were sensitive to X-ray irradiation at room temperature, the frozen crystals at 100 K showed no apparent decay during the intensity measurement.

Characterization of the oligomeric states of RecA protein: monomeric RecA protein can form a nucleoprotein filament.

Self-assembly of RecA protein in solution and on single-stranded DNA exerts a significant effect on the catalytic activities of this protein. To manipulate the self-association reaction, we examined the effects of various salts on the self-association of RecA from Thermus thermophilus (ttRecA) by circular dichroism spectroscopy and gel-filtration analysis. We showed that the self-association of ttRecA strongly depends on the kind and concentration of the salt, as well as on the protein concentration. Chaotropic ions were especially useful for obtaining RecA in its hexameric and monomeric states. On the basis of these observations, we were able to regulate the oligomeric states of ttRecA and we then examined the activity of RecA in various oligomeric states. Monomeric ttRecA bound to ssDNA and formed a nucleoprotein filament, which showed ssDNA-dependent ATPase activity. These results suggest that the monomeric form of RecA is an intermediate in filament formation on ssDNA.

Domain organization and functional analysis of Thermus thermophilus MutS protein.

MutS protein binds to DNA and specifically recognizes mismatched or small looped out heteroduplex DNA. In order to elucidate its structure-function relationships, the domain structure of Thermus thermophilus MutS protein was studied by performing denaturation experiments and limited proteolysis. The former suggested that T. thermophilus MutS consists of at least three domains with estimated stabilities of 12.3, 22.9 and 30.7 kcal/mol and the latter revealed that it consists of four domains: A1 (N-terminus to residue 130), A2 (131-274), B (275-570) and C (571 to C-terminus). A gel retardation assay indicated that T.thermophilus MutS interacts non-specifically with double-stranded (ds), but not single-stranded DNA. Among the proteolytic fragments, the B domain bound to dsDNA. On the basis of these results we have proposed the domain organization of T. thermophilus MutS and putative roles of these domains.

Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid.

RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.

RecA protein has extremely high cooperativity for substrate in its ATPase activity.

The single-stranded DNA-dependent ATPase activity of Escherichia coli RecA protein, especially its cooperativity for ATP, was investigated. To measure the ATPase activity in detail, the methods and reaction conditions for the ATPase assay were reexamined. Under conditions where RecA protein always showed a maximal rate of ATP hydrolysis, its poly(dT)-dependent ATPase activity was measured. At 25 degrees C, increasing the concentration of RecA protein from 0.3 to 1.0 microM increased the turnover number (kcat) from 0.16 to 0.19 s-1 and the Hill coefficient (nH) for ATP from 9.3 to 11.6. At 0.5 microM RecA protein, increasing the temperature from 25 to 37 degrees C increased kcat from 0.18 to 0.35 s-1 but decreased nH from 9.8 to 6.6. Interestingly, the ATPase activity of RecA protein measured in this study showed much higher cooperativity for ATP than those reported to date. Furthermore, the nH value of 11.6 for ATP obtained here was the highest of any ATPase reported so far. These results suggest that the binding of an ATP molecule to a RecA molecule within a nucleoprotein helical filament causes structural change of many other neighboring RecA molecules. This implies that ATP binding induces structural change of the whole nucleoprotein helical filament. Finally, we demonstrated that analysis of cooperativity is useful for revealing how a protein composed of many subunits functions as a whole.

Local folding of the N-terminal domain of Escherichia coli RecA controls protein-protein interaction.

To obtain structural information about the self-association of the protein RecA, we studied urea denaturation of RecA by circular dichroism spectroscopy and gel filtration. Gel filtration analysis showed that urea at low concentrations, 1.0-1.2 M, dissociated the RecA oligomer to almost a monomeric state prior to the unfolding of each molecule. Upon treatment with 1.0 M urea, the circular dichroism spectrum showed a decrease in the alpha-helical content of RecA. A similar decrease was observed in the absence of urea for RecA at an extremely low protein concentration; the RecA oligomer dissociated to an almost completely monomeric state. The properties of RecA at low urea concentrations were similar to those of a truncated RecA lacking the first 33 N-terminal residues (Delta33RecA). Addition of a synthetic peptide corresponding to the 33 N-terminal residues to Delta33RecA increased the alpha-helical content. These results suggest that local folding of the N-terminal domain is coupled to protein-protein interactions of monomeric RecA, which are involved in the regulation of filament formation. The dissociation constant for interaction between RecA monomers was determined from the ellipticity data to be 0.1 microM.