Structure and Function of the Small MutS-Related Domain

MutS family proteins are widely distributed in almost all organisms from bacteria to human and play central roles in various DNA transactions such as DNA mismatch repair and recombinational events. The small MutS-related (Smr) domain was originally found in the C-terminal domain of an antirecombination protein, MutS2, a member of the MutS family. MutS2 is thought to suppress homologous recombination by endonucleolytic resolution of early intermediates in the process. The endonuclease activity of MutS2 is derived from the Smr domain. Interestingly, sequences homologous to the Smr domain are abundant in a variety of proteins other than MutS2 and can be classified into 3 subfamilies. Recently, the tertiary structures and endonuclease activities of all 3 Smr subfamilies were reported. In this paper, we review the biochemical characteristics and structures of the Smr domains as well as cellular functions of the Smr-containing proteins

Inactivation of the DNA Repair Genes mutS, mutL or the Anti-Recombination Gene mutS2 Leads to Activation of Vitamin B1 Biosynthesis Genes

Oxidative stress generates harmful reactive oxygen species (ROS) that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B1, which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system

Analysis of a nuclease activity of catalytic domain of Thermus thermophilus MutS2 by high-accuracy mass spectrometry

Electrospray ionization with Fourier-transform ion cyclotron resonance mass spectrometry (ESI–FT ICR MS) is a powerful tool for analyzing the precise structural features of biopolymers, including oligonucleotides. Here, we described the detailed characterization of a newly discovered nuclease activity of the C-terminal domain of Thermus thermophilus MutS2 (ttMutS2). Using this method, the length, nucleotide content and nature of the 5′- and 3′-termini of the product oligonucleotides were accurately identified. It is revealed that the C-terminal domain of ttMutS2 incised the phosphate backbone of oligodeoxynucleotides non-sequence-specifically at the 3′ side of the phosphates. The simultaneous identification of the innumerable fragments was achieved by the extremely high-accuracy of ESI–FT ICR MS

The structure of TTHA0988 from Thermus thermophilus, a KipI-KipA homologue incorrectly annotated as allophanate hydrolase

The Thermus thermophilus protein TTHA0988 is a protein of unknown function which represents a fusion of two proteins found almost ubiquitously across the bacterial kingdom. These two proteins perform a role regulating sporulation in Bacillus subtilis, where they are known as KipI and KipA. kipI and kipA genes are usually found immediately adjacent to each other and are often fused to produce a single polypeptide, as is the case with TTHA0988. Here, three crystal forms are reported of TTHA0988, the first structure to be solved from the family of `KipI-KipA fusion' proteins. Comparison of the three forms reveals structural flexibility which can be described as a hinge motion between the `KipI' and `KipA' components. TTHA0988 is annotated in various databases as a putative allophanate hydrolase. However, no such activity could be identified and genetic analysis across species with known allophanate hydrolases indicates that a misannotation has occurred. © 2011, Wiley-Blackwell. The definitive version is available at www3.interscience.wiley.co

Purification and Characterization of Cystathionine γ-Synthase from Thermoacidophilic Archaea Sulfolobus tokodaii

The gene encoding a cystathionine γ-synthase from Sulfolobus tokodaii was cloned and expressed in Escherihia coli Rosetta-gami (DE3). Cystathionine γ-synthase ［EC 2. 5. 1. 48］ from Sulfolobus tokodaii (stCGS) was purified by heat treatment, DEAE- Toyopearl 650M and Sephacryl S-300 column chromatographies from E. coli transformants. stCGS shows optimum activity at pH 7.0, and is stable between pH5.0 and pH9.0. The optimum temperature of stCGS is above 100℃, and the enzyme showed the remaining activity of almost 100% up to 70℃. The K(m) and V(max) with O-phospho-L- homoserine as a substrate are 0.82 mM and 2.42 U/mg. To analyze the role of Phe 97 in the active site of stCGS, we constructed F97Y, R99C, and F97Y-R99C mutant enzymes. Although native stCGS has no activity toward l-methionine, F97Y mutant enzyme gained the elimination activity toward L-methionine.好熱好酸性アーキア Sulfolobus tokodaii 由来シスタチオニンγﾝシンターゼ（stCGS）遺伝子を pET-11a に組み込み pET-stCGS を構築した．このベクターでE. coli Rosettaﾝgami（DE3）を形質転換し，本遺伝子を発現させ，精製及び性質検討を行った．大腸菌で発現したシスタチオニンγﾝシンターゼの活性が無細胞抽出液で確認できた．S. tokodaii シスタチオニンγﾝシンターゼを70℃熱処理 DEAEﾝトヨパールイオン交換カラム等により単一精製した．精製酵素の最適温度は100℃以上であり，熱安定性は60分間処理で70℃までほぼ100ｵの残存活性を示した．また，最適pHについてはリン酸緩衝液やブリトンﾝロビンソン広域緩衝液の場合はpH7.0の時が最も活性が高く，トリス塩酸緩衝液の場合はpH9.0が最適であった．pH安定性についてはpH5.0～9.0において安定であった．O-ホスホ-l-ホモセリンに対するKm，Vmaxは，それぞれ0.82mM，2.42U/㎎であった．アポ酵素のホロ化実験により，本酵素活性がPLP に依存していることが明らかとなった．更に本酵素の脱離反応での基質特異性の検討を行った．変異酵素を用いた実験により，stCGSの基質特異性には，活性中心に存在するPhe97を含む領域が深く関わっていることが示唆された

The role of ribonucleases in regulating global mRNA levels in the model organism Thermus thermophilus HB8

BACKGROUND: RNA metabolism, including RNA synthesis and RNA degradation, is one of the most conserved biological systems and has been intensively studied; however, the degradation network of ribonucleases (RNases) and RNA substrates is not fully understood. RESULTS: The genome of the extreme thermophile, Thermus thermophilus HB8 includes 15 genes that encode RNases or putative RNases. Using DNA microarray analyses, we examined the effects of disruption of each RNase on mRNA abundance. Disruption of the genes encoding RNase J, RecJ-like protein and RNase P could not be isolated, indicating that these RNases are essential for cell viability. Disruption of the TTHA0252 gene, which was not previously considered to be involved in mRNA degradation, affected mRNA abundance, as did disruption of the putative RNases, YbeY and PhoH-like proteins, suggesting that they have RNase activity. The effects on mRNA abundance of disruption of several RNase genes were dependent on the phase of cell growth. Disruption of the RNase Y and RNase HII genes affected mRNA levels only during the log phase, whereas disruption of the PhoH-like gene affected mRNA levels only during the stationary phase. Moreover, disruption of the RNase R and PNPase genes had a greater impact on mRNA abundance during the stationary phase than the log phase, whereas the opposite was true for the TTHA0252 gene disruptant. Similar changes in mRNA levels were observed after disruption of YbeY or PhoH-like genes. The changes in mRNA levels in the bacterial Argonaute disruptant were similar to those in the RNase HI and RNase HII gene disruptants, suggesting that bacterial Argonaute is a functional homolog of RNase H. CONCLUSION: This study suggests that T. thermophilus HB8 has 13 functional RNases and that each RNase has a different function in the cell. The putative RNases, TTHA0252, YbeY and PhoH-like proteins, are suggested to have RNase activity and to be involved in mRNA degradation. In addition, PhoH-like and YbeY proteins may act cooperatively in the stationary phase. This study also suggests that endo-RNases function mainly during the log phase, whereas exo-RNases function mainly during the stationary phase. RNase HI and RNase HII may have similar substrate selectivity

Entropic stabilization of the tryptophan synthase α-subunit from a hyperthermophile, Pyrococcus furiosus : X-ray analysis and calorimetry

This research was originally published in Journal of Biological Chemistry. Yuriko Yamagata, Kyoko Ogasahara, Yusaku Hioki, Soo Jae Lee, Atsushi Nakagawa, Haruki Nakamura, Masami Ishida, Seiki Kuramitsui, and Katsuhide Yutani. Entropic stabilization of the tryptophan synthase α-subunit from a hyperthermophile, Pyrococcus furiosus : X-ray analysis and calorimetry. J. Biol. Chem. 2001; 276, 11062-11071. © the American Society for Biochemistry and Molecular Biology

Molecular Mechanisms of the Whole DNA Repair System: A Comparison of Bacterial and Eukaryotic Systems

DNA is subjected to many endogenous and exogenous damages. All organisms have developed a complex network of DNA repair mechanisms. A variety of different DNA repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. Recent studies of the fundamental mechanisms for DNA repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as functional interactions between proteins involved in repair pathways. In this paper we give a broad overview of the whole DNA repair system and focus on the molecular basis of the repair machineries, particularly in Thermus thermophilus HB8

Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

SummaryWe developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation