Identification of a radical SAM enzyme involved in the synthesis of archaeosine.

Archaeosine (G+), 7-formamidino-7-deazaguanosine, is an archaea-specific modified nucleoside found at the 15th position of tRNAs. In Euryarchaeota, 7-cyano-7-deazaguanine (preQ0)-containing tRNA (q0N-tRNA), synthesized by archaeal tRNA-guanine transglycosylase (ArcTGT), has been believed to be converted to G+-containing tRNA (G+-tRNA) by the paralog of ArcTGT, ArcS. However, we found that several euryarchaeal ArcSs have lysine transfer activity to q0N-tRNA to form q0kN-tRNA, which has a preQ0 lysine adduct as a base. Through comparative genomics and biochemical experiments, we found that ArcS forms a robust complex with a radical S-adenosylmethionine (SAM) enzyme named RaSEA. The ArcS-RaSEA complex anaerobically converted q0N-tRNA to G+-tRNA in the presence of SAM and lysine via q0kN-tRNA. We propose that ArcS and RaSEA should be considered an archaeosine synthase α-subunit (lysine transferase) and ß-subunit (q0kN-tRNA lyase), respectively.

The Cdc45/RecJ-like protein forms a complex with GINS and MCM, and is important for DNA replication in Thermococcus kodakarensis.

The archaeal minichromosome maintenance (MCM) has DNA helicase activity, which is stimulated by GINS in several archaea. In the eukaryotic replicative helicase complex, Cdc45 forms a complex with MCM and GINS, named as CMG (Cdc45-MCM-GINS). Cdc45 shares sequence similarity with bacterial RecJ. A Cdc45/RecJ-like protein from Thermococcus kodakarensis shows a bacterial RecJ-like exonuclease activity, which is stimulated by GINS in vitro. Therefore, this archaeal Cdc45/RecJ is designated as GAN, from GINS-associated nuclease. In this study, we identified the CMG-like complex in T. kodakarensis cells. The GAN·GINS complex stimulated the MCM helicase, but MCM did not affect the nuclease activity of GAN in vitro. The gene disruption analysis showed that GAN was non-essential for its viability but the Δgan mutant did not grow at 93°C. Furthermore, the Δgan mutant showed a clear retardation in growth as compared with the parent cells under optimal conditions at 85°C. These deficiencies were recovered by introducing the gan gene encoding the nuclease deficient GAN protein back to the genome. These results suggest that the replicative helicase complex without GAN may become unstable and ineffective in replication fork progression. The nuclease activity of GAN is not related to the growth defects of the Δgan mutant cells.

The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3'-5' exonuclease that associates with a DNA replication complex.

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e. in bacteria, Eukarya, and Archaea. Bacterial RecJ is a 5'-3' exonuclease and functions in DNA repair pathways by using its 5'-3' exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity but participates in the CMG complex, so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11-3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was, therefore, designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ·MCM·GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ-like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5'-3' exonuclease activity, whereas TaRecJ2 had 3'-5' exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2·TaGINS complex stimulated activity of TaMCM (T. acidophilum MCM) helicase in vitro, and the TaRecJ2·TaMCM·TaGINS complex was also observed in vivo However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya.

Atomic structure of an archaeal GAN suggests its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication.

In eukaryotic DNA replication initiation, hexameric MCM (mini-chromosome maintenance) unwinds the template double-stranded DNA to form the replication fork. MCM is activated by two proteins, Cdc45 and GINS, which constitute the 'CMG' unwindosome complex together with the MCM core. The archaeal DNA replication system is quite similar to that of eukaryotes, but only limited knowledge about the DNA unwinding mechanism is available, from a structural point of view. Here, we describe the crystal structure of an archaeal GAN (GINS-associated nuclease) from Thermococcus kodakaraensis, the homolog of eukaryotic Cdc45, in both the free form and the complex with the C-terminal domain of the cognate Gins51 subunit (Gins51C). This first archaeal GAN structure exhibits a unique, 'hybrid' structure between the bacterial RecJ and the eukaryotic Cdc45. GAN possesses the conserved DHH and DHH1 domains responsible for the exonuclease activity, and an inserted CID (CMG interacting domain)-like domain structurally comparable to that in Cdc45, suggesting its dual roles as an exonuclease in DNA repair and a CMG component in DNA replication. A structural comparison of the GAN-Gins51C complex with the GINS tetramer suggests that GINS uses the mobile Gins51C as a hook to bind GAN for CMG formation.

Disordered interdomain region of Gins is important for functional tetramer formation to stimulate MCM helicase in Thermoplasma acidophilum.

The eukaryotic MCM is activated by forming the CMG complex with Cdc45 and GINS to work as a replicative helicase. The eukaryotic GINS consists of four different proteins to form tetrameric complex. In contrast, the TaGins51 protein from the thermophilic archaeon, Thermoplasma acidophilum forms a homotetramer (TaGINS), and interacts with the cognate MCM (TaMCM) to stimulate the DNA-binding, ATPase, and helicase activities of TaMCM. All Gins proteins from Archaea and Eukarya contain α-helical A- and ß-stranded B-domains. Here, we found that TaGins51 forms the tetramer without the B-domain. However, the A-domain without the linker region between the A- and B-domains could not form a stable tetramer, and furthermore, the A-domain by itself could not stimulate the TaMCM activity. These results suggest that the formation of the Gins51 tetramer is necessary for MCM activation, and the disordered linker region between the two domains is critical for the functional complex formation.

Activation of the MCM helicase from the thermophilic archaeon, Thermoplasma acidophilum by interactions with GINS and Cdc6-2.

In DNA replication studies, the mechanism for regulation of the various steps from initiation to elongation is a crucial subject to understand cell cycle control. The eukaryotic minichromosome maintenance (MCM) protein complex is recruited to the replication origin by Cdc6 and Cdt1 to form the pre-replication complex, and participates in forming the CMG complex formation with Cdc45 and GINS to work as the active helicase. Intriguingly, Thermoplasma acidophilum, as well as many other archaea, has only one Gins protein homolog, contrary to the heterotetramer of the eukaryotic GINS made of four different proteins. The Gins51 protein reportedly forms a homotetramer (TaGINS) and physically interacts with TaMCM. In addition, TaCdc6-2, one of the two Cdc6/Orc1 homologs in T. acidophilum reportedly stimulates the ATPase and helicase activities of TaMCM in vitro. Here, we found a reaction condition, in which TaGINS stimulated the ATPase and helicase activities of TaMCM in a concentration dependent manner. Furthermore, the stimulation of the TaMCM helicase activity by TaGINS was enhanced by the addition of TaCdc6-2. A gel retardation assay revealed that TaMCM, TaGINS, and TaCdc6-2 form a complex on ssDNA. However, glutaraldehyde-crosslinking was necessary to detect the shifted band, indicating that the ternary complex of TaMCM-TaGINS-TaCdc6-2 is not stable in vitro. Immunoprecipitation experiment supported a weak interaction of these three proteins in vivo. Activation of the replicative helicase by a mechanism including a Cdc6-like protein suggests the divergent evolution after the division into Archaea and Eukarya.

Biochemical and genetical analyses of the three mcm genes from the hyperthermophilic archaeon, Thermococcus kodakarensis.

In eukaryotes, the replicative DNA helicase 'core' is the minichromosome maintenance (Mcm) complex (MCM), forming a heterohexameric complex consisting of six subunits (Mcm2-7). Recent studies showed that the CMG (Cdc45-MCM-GINS) complex is the actual helicase body in the replication fork progression complex. In Archaea, Thermococcus kodakarensis harbors three genes encoding the Mcm homologs on its genome, contrary to most archaea, which have only one homolog. It is thus, of high interest, whether and how these three Mcms share their functions in DNA metabolism in this hyperthermophile. Here, we report the biochemical properties of two of these proteins, TkoMcm1 and TkoMcm3. In addition, their physical and functional interactions with GINS, possibly an essential factor for the initiation and elongation process of DNA replication, are presented through in vitro ATPase and helicase assays, and an in vivo immunoprecipitation assay. Gene disruption and product quantification analyses suggested that TkoMcm3 is essential for cell growth and plays a key role as the main DNA helicase in DNA replication, whereas TkoMcm1 also shares some function in the cells.

Architectures of archaeal GINS complexes, essential DNA replication initiation factors.

BACKGROUND: In the early stage of eukaryotic DNA replication, the template DNA is unwound by the MCM helicase, which is activated by forming a complex with the Cdc45 and GINS proteins. The eukaryotic GINS forms a heterotetramer, comprising four types of subunits. On the other hand, the archaeal GINS appears to be either a tetramer formed by two types of subunits in a 2:2 ratio (α2ß2) or a homotetramer of a single subunit (α4). Due to the low sequence similarity between the archaeal and eukaryotic GINS subunits, the atomic structures of the archaeal GINS complexes are attracting interest for comparisons of their subunit architectures and organization. RESULTS: We determined the crystal structure of the α2ß2 GINS tetramer from Thermococcus kodakaraensis (TkoGINS), comprising Gins51 and Gins23, and compared it with the reported human GINS structures. The backbone structure of each subunit and the tetrameric assembly are similar to those of human GINS. However, the location of the C-terminal small domain of Gins51 is remarkably different between the archaeal and human GINS structures. In addition, TkoGINS exhibits different subunit contacts from those in human GINS, as a consequence of the different relative locations and orientations between the domains. Based on the GINS crystal structures, we built a homology model of the putative homotetrameric GINS from Thermoplasma acidophilum (TacGINS). Importantly, we propose that a long insertion loop allows the differential positioning of the C-terminal domains and, as a consequence, exclusively leads to the formation of an asymmetric homotetramer rather than a symmetrical one. CONCLUSIONS: The DNA metabolizing proteins from archaea are similar to those from eukaryotes, and the archaeal multi-subunit complexes are occasionally simplified versions of the eukaryotic ones. The overall similarity in the architectures between the archaeal and eukaryotic GINS complexes suggests that the GINS function, directed through interactions with other protein components, is basically conserved. On the other hand, the different subunit contacts, including the locations and contributions of the C-terminal domains to the tetramer formation, imply the possibility that the archaeal and eukaryotic GINS complexes contribute to DNA unwinding reactions by significantly different mechanisms in terms of the atomic details.

The GINS complex from the thermophilic archaeon, Thermoplasma acidophilum may function as a homotetramer in DNA replication.

The eukaryotic GINS heterotetramer, consisting of Sld5, Psf1, Psf2, and Psf3, participates in "CMG complex" formation with mini-chromosome maintenance (MCM) and Cdc45 as a key component of a replicative helicase. There are only two homologs of the GINS proteins in Archaea, and these proteins, Gins51 and Gins23, form a heterotetrameric GINS with a 2:2 molar ratio. The Pyrococcus furiosus GINS stimulates the ATPase and helicase activities of its cognate MCM, whereas the Sulfolobus solfataricus GINS does not affect those activities of its cognate MCM, although the proteins bind each other. Intriguingly, Thermoplasma acidophilum, as well as many euryarchaea, have only one gene encoding the sequence homologous to that of archaeal Gins protein (Gins51) on the genome. In this study, we investigated the biochemical properties of the gene product (TaGins51). A gel filtration and electron microscopy revealed that TaGins51 forms a homotetramer. A physical interaction between TaGins51 and TaMcm was detected by a surface plasmon resonance analysis. Unexpectedly, TaGins51 inhibited the ATPase activity, but did not affect the helicase activity of its cognate MCM. These results suggest that another factor is required to form a stable helicase complex with MCM and GINS at the replication fork in T. acidophilum cells.

In vivo and in vitro effects of fluoroquinolones on lipopolysaccharide-induced pro-inflammatory cytokine production.

Fluoroquinolones have been reported to affect cytokine production in vitro. We investigated the effects of fluoroquinolones on lipopolysaccharide (LPS)-induced inflammatory cytokine production in vivo and in vitro. LPS was administered to mice treated with ciprofloxacin, gatifloxacin, norfloxacin, and levofloxacin, and the serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 6 (IL-6) were measured. In addition, peritoneal macrophages collected from mice were treated with the four fluoroquinolones for 1 h, followed by the addition of LPS, and the TNF-alpha, IL-1beta, and IL-6 levels in culture fluid were measured. In LPS-treated mice, ciprofloxacin, gatifloxacin, and norfloxacin (100 mg/kg) significantly reduced the serum TNF-alpha level (6.8%-63.6% of control). Levofloxacin at 100 mg/kg did not affect the TNF-alpha level, whereas levofloxacin at a lower dose (10 mg/kg) significantly increased the level. All four fluoroquinolones (100 mg/kg) investigated in this study tended to decrease the serum IL-1beta levels (65.5%-65.9% of control), but this was not a significant change. The serum IL-6 levels were increased in ciprofloxacin-administered mice, whereas the other fluoroquinolones did not affect the serum IL-6 levels. In mouse peritoneal macrophages, LPS induced TNF-alpha, IL-1beta, and IL-6 production. Ciprofloxacin, gatifloxacin, and norfloxacin (100 mug/ml) inhibited both TNF-alpha (12.1%-69.0% of control) and IL-1beta production (22.1%-68.8% of control). Levofloxacin (100 mug/ml) inhibited IL-1beta production (65.0% of control), but not TNF-alpha production. LPSstimulated IL-6 production was inhibited only by norfloxacin (59.5 % of control). Our in vivo and in vitro results suggest that fluoroquinolones, especially ciprofloxacin, gatifloxacin, and norfloxacin, which have a cyclopropyl group at the N1 position and/or a piperazinyl group at the C7 position, modify inflammatory responses.

Fluoroquinolones reduce carrageenan-induced edema in rats and the involvement of the glucocorticoid receptor system.

We studied the effect of fluoroquinolones (FQs) on carrageenan-induced edema in the rat footpad. Ciprofloxacin, gatifloxacin, sparfloxacin, norfloxacin, and enoxacin (s.c., 100 mg/kg), which have piperazinyl and/or cyclopropyl groups, inhibited carrageenan-induced edema, whereas levofloxacin, tosufloxacin, and pazufloxacin did not. The reduction of edema by ciprofloxacin, sparfloxacin, and enoxacin was abolished by pretreatment with mifepristone, an antagonist of the glucocorticoid receptor. These results suggest that FQs with piperazinyl and/or cyclopropyl groups can modify biological responses through enhancing the glucocorticoid-glucocorticoid receptor system.

Photochange in pore diameters of azobenzene-planted mesoporous silica materials.

Azobenzene (Az) groups were planted on the pore wall of mesoporous silica MCM-41 (M41) by silylation of triethoxy[4-phenylazo(phenyl)]silane. The optimal surface density of Az groups was 0.9 group nm-2, and too much loading of Az induced the lowering of the efficiency of the trans-cis isomerization due to the congestion of the groups. The reversible change in the pore diameters upon UV-vis irradiation could not be confirmed by N2 adsorption at 77 K but was revealed to be ca. 1.0 nm by the shift of the UV-vis absorption band of p-N,N-dimethylaminobenzylidenemalononitrile introduced into the Az-modified pores.