Ryuichi Kato

Crystal structure of a repair enzyme of oxidatively damaged DNA. MutM (FPG), from an extreme thermophile, Thermus thermophilus HB8

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N‐glycosylase activity) and cleaves both the 3′‐ and 5′‐phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 Å resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn2+ ion of the zinc finger. MutM is composed of two distinct and novel domains connected by a flexible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three‐dimensional structure and taking account of previous biochemical experiments, we propose a DNA‐binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA‐binding motifs (the zinc finger and the helix–two‐turns–helix motifs) suggest that the oxidized base is flipped out from double‐stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes.

Physical interactions between DinI and RecA nucleoprotein filament for the regulation of SOS mutagenesis.

The Escherichia coli dinI gene is one of the LexA‐regulated genes, which are induced upon DNA damage. Its overexpression conferred severe UV sensitivity on wild‐type cells and resulted in the inhibition of LexA and UmuD processing, reactions that are normally dependent on activated RecA in a complex with single‐stranded (ss)DNA. Here, we study the mechanism by which DinI inhibits the activities of RecA. While DinI neither binds to ssDNA nor prevents the formation of RecA nucleoprotein filament, it binds to active RecA filament, thereby inhibiting its coprotease activity but not the ATPase activity. Furthermore, even under in vitro conditions where UmuD cleavage dependent on RecA–ssDNA–adeno sine‐5′‐(3‐thiotriphosphate) is blocked in the presence of DinI, LexA is cleaved normally. This result, taken together with electron microscopy observations and linear dichroism measurements, indicates that the ternary complex remains intact in the presence of DinI, and that the affinity to the RecA filament decreases in the order LexA, DinI and UmuD. DinI is thus suited to modulating UmuD processing so as to limit SOS mutagenesis.

Domain Structure of Thermus thermophilus UvrB Protein SIMILARITY IN DOMAIN STRUCTURE TO A HELICASE

UvrB protein plays an essential role in the prokaryotic excision repair system. UvrB protein shows cryptic ATPase activity, DNA binding, helicase-like activity, and incision activity by interacting with UvrA or UvrC proteins. To reveal the structure-function relationship of this multifunctional protein, the domain structure of Thermus thermophilus UvrB protein (ttUvrB) was studied by limited proteolysis and denaturation experiments. Proteolytic profiles indicated that ttUvrB consists of four domains: the N domain (residues 2–105), M domain (106–455), C1 domain (456–590), and C2 domain (591–665). The properties of the proteolytic fragments indicated the involvement of the respective domains in the functions of the protein as follows: the N and C1 domains are necessary for ATPase activity, the C1 domain is indispensable for DNA binding, and the N and/or M domains are involved in UvrA binding. The structural stability of the C1 and C2 domains was higher than that of the N and M domains, which supports the proposed domain nature of ttUvrB. Based on these results and the crystal structure of PcrA helicase (Subramanya, H. S., Bird, L. E., Brannigan, J. A., and Wigley, D. B. (1996) Nature384, 379–383), the domain organization of ttUvrB was proposed.

Cloning, sequencing and expression of the uvrA gene from an extremely thermophilic bacterium, Thermus thermophilus HB8.

One of the most important DNA repair systems is the nucleotide (nt) excision repair system. The uvr A gene, which plays an essential role in the prokaryotic excision repair system, was cloned from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) HB8, and its nt sequence was determined. In the amino acid (aa) sequence of Tt UvrA, a characteristic duplicated structure, two nt-binding consensus sequences (Walkers A-type motif) and two zinc finger DNA-binding motifs were found. The aa sequence showed 73% homology with that of Escherichia coli (Ec). These features suggest that Tt has the same excision repair system as Ec. Upon comparison of the Tt and Ec UvrA, some characteristic aa substitutions were found. The numbers of Arg and Pro residues were increased (from 66 to 81 and from 47 to 55, respectively), and the numbers of Asn and Met residues were decreased (from 33 to 18 and from 18 to 11, respectively) in Tt. The Tt uvr A gene was expressed in Ec under control of the lac promoter. Purified UvrA was stable up to 80 degrees C (at neutral pH) and at pH 2-11 (at 25 degrees C).

DNA binding and protein-protein interaction sites in MutS, a mismatched DNA recognition protein from Thermus thermophilus HB8.

The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest thatThermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998)Nucleic Acids Res. 26, 4153–4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced inEscherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walkers A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.

Cloning and characterization of the uvrD gene from an extremely thermophilic bacterium, Thermus thermophilus HB8

The uvrD gene encodes a DNA helicase which plays an important role in prokaryotic nucleotide (nt) excision repair, mismatch repair and DNA replication. A cosmid-based genomic DNA library for Thermus thermophilus (Tt) HB8 was constructed, and this was screened by Southern hybridization using a uvrD fragment amplified by PCR as the probe. The nt sequence of cloned Tt uvrD was then determined. Characteristic helicase motifs, made up of seven elements, were all conserved in the amino acid (aa) sequence of Tt UvrD. The aa sequence showed 41% homology with that of Escherichia coli (Ec). In the aa composition of Tt UvrD, the number of Asn, Gln, Met and Cys residues was decreased, and the number of Pro residues was increased. The distribution of Pro residues and recent data on X-ray crystallographic structure suggested the importance of the structural dynamics of the protein. These changes are thought to stabilize the native protein conformation against heat denaturation. Tt uvrD complemented the UV sensitivity of a Ec uvrD mutant. Thus, the thermophilic bacterium has a UvrD helicase, whose function is common to Ec UvrD.

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.

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 Å. The value of R g decreased to 36 Å 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.

Crystallization and preliminary X-ray characterization of aspartate aminotransferase from an extreme thermophile, Thermus thermophilus HB8

Recombinant aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8, has been crystallized in two different crystal forms. The crystals of both forms are orthorhombic and belong to space group P212121 with cell dimensions a = 124.3, b = 113.6 and c = 61.6 A for form I and a = 197.3, b = 109.7 and c = 80.3 A for form II. The crystals of form I and II diffract to 2.1 and 2.5 A resolution, respectively, on a conventional laboratory rotating-anode source. Two heavy-atom derivatives have been identified for form I.