Role of RNase H1 in DNA repair: removal of single ribonucleotide misincorporated into DNA in collaboration with RNase H2.

Several RNases H1 cleave the RNA-DNA junction of Okazaki fragment-like RNA-DNA/DNA substrate. This activity, termed 3'-junction ribonuclease (3'-JRNase) activity, is different from the 5'-JRNase activity of RNase H2 that cleaves the 5'-side of the ribonucleotide of the RNA-DNA junction and is required to initiate the ribonucleotide excision repair pathway. To examine whether RNase H1 exhibits 3'-JRNase activity for dsDNA containing a single ribonucleotide and can remove this ribonucleotide in collaboration with RNase H2, cleavage of a DNA8-RNA1-DNA9/DNA18 substrate with E.

Structure, activity, and stability of metagenome-derived glycoside hydrolase family 9 endoglucanase with an N-terminal Ig-like domain.

A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9.

Structure and stability of metagenome-derived glycoside hydrolase family 12 cellulase (LC-CelA) a homolog of Cel12A from Rhodothermus marinus.

Ten genes encoding novel cellulases with putative signal peptides at the N-terminus, termed pre-LC-CelA-J, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using agar plates containing carboxymethyl cellulose and trypan blue. All the cellulases except pre-LC-CelG have a 14-29 residue long flexible linker (FL) between the signal peptide and the catalytic domain. LC-CelA without a signal peptide (residues 20-261), which shows 76% amino acid sequence identity to Cel12A from Rhodothermus marinus (RmCel12A), was overproduced in Escherichia coli, purified and characterized.

Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1∼6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%.

Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.

The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase.

Crystal structure of metagenome-derived LC9-RNase H1 with atypical DEDN active site motif.

The crystal structure of metagenome-derived LC9-RNase H1 was determined. The structure-based mutational analyses indicated that the active site motif of LC9-RNase H1 is altered from DEDD to DEDN. In this motif, the location of the second glutamate residue is moved from αA-helix to β1-strand immediately next to the first aspartate residue, as in the active site of RNase H2.

Crystal structure of metagenome-derived LC11-RNase H1 in complex with RNA/DNA hybrid.

LC11-RNase H1 is a Sulfolobus tokodaii RNase H1 (Sto-RNase H1) homologue isolated by metagenomic approach. In this study, the crystal structure of LC11-RNase H1 in complex with an RNA/DNA substrate was determined. Unlike Bacillus halodurans RNase H1 without hybrid binding domain (HBD) (Bh-RNase HC) and human RNase H1 without HBD (Hs-RNase HC), LC11-RNase H1 interacts with four non-consecutive 2'-OH groups of the RNA strand.

Structure and characterization of RNase H3 from Aquifex aeolicus.

The crystal structure of ribonuclease H3 from Aquifex aeolicus (Aae-RNase H3) was determined at 2.0 Å resolution. Aae-RNase H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase H domain (C-domain).

Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.

Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA).

Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach.

The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase.

Flavobacterium compostarboris sp. nov., isolated from leaf-and-branch compost, and emended descriptions of Flavobacterium hercynium, Flavobacterium resistens and Flavobacterium johnsoniae.

A strictly aerobic, Gram-negative, yellow-pigmented, non-spore-forming rod, designated 15C3(T), was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. Growth was observed at 9-33 °C (optimum 25 °C) and pH 5.6-7.

The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.

Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD.

Flavobacterium banpakuense sp. nov., isolated from leaf-and-branch compost.

A strictly aerobic, Gram-stain-negative, yellow-pigmented, non-spore-forming, motile (by gliding), rod-shaped bacterium, designated strain 15F3(T), was isolated from leaf-and-branch compost. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15F3(T) was most closely related to Flavobacterium reichenbachii WB 3.2-61(T) and formed a distinct phyletic lineage within the genus Flavobacterium, the type genus of the family Flavobacteriaceae.

The region of CQQQKPQRRP of PGC-1alpha interacts with the DNA-binding complex of FXR/RXRalpha.

PGC-1alpha co-activates transcription by several nuclear receptors. To study the interaction among PGC-1alpha, RXRalpha/FXR, and DNA, we performed electrophoresis mobility shift assays. The RXRalpha/FXR proteins specifically bound to DNA containing the IR-1 sequence in the absence of ligand.

The nuclear bile acid receptor FXR is activated by PGC-1alpha in a ligand-dependent manner.

The nuclear bile acid receptor FXR (farnesoid X receptor) is one of the key factors that suppress bile acid biosynthesis in the liver. PGC-1alpha [PPARgamma (peroxisome-proliferator-activated receptor gamma) co-activator-1alpha] is known to control energy homoeostasis in adipose tissue, skeletal muscle and liver. We performed cell-based reporter assays using the expression system of a GAL4-FXR chimaera, the ligand-binding domain of FXR fused to the DNA-binding domain of yeast GAL4, to find the co-activators for FXR.

Activation of orphan nuclear constitutive androstane receptor requires subnuclear targeting by peroxisome proliferator-activated receptor gamma coactivator-1 alpha. A possible link between xenobiotic response and nutritional state.

In contrast to the classical nuclear receptors, the constitutive androstane receptor (CAR) is transcriptionally active in the absence of ligand. In the course of searching for the mediator of CAR activation, we found that ligand-independent activation of CAR was achieved in cooperation with the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha). PGC-1 beta, a PGC-1 alpha homologue, also activated CAR to less of an extent than PGC-1 alpha.

Non-sequence-specific DNA binding by the FILAMENTOUS FLOWER protein from Arabidopsis thaliana is reduced by EDTA.

The FILAMENTOUS FLOWER protein has a zinc finger domain, hydrophobic region, proline-rich region, and a HMG box-like domain. We have reported that zinc release at the zinc finger is probably facilitated by the non-canonical cysteine residue at position 56, and that EDTA causes the structural change and enhances the self-assembly of the protein (Kanaya, E., Watanabe, K.