The bacterial Mre11-Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions.

The Mre11-Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11-Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3'-5' exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates.

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.

The HerA-NurA helicase-nuclease complex cooperates with Mre11 and Rad50 to coordinate the repair of double-stranded DNA breaks. Little is known, however, about the assembly mechanism and activation of the HerA-NurA. By combining hybrid mass spectrometry with cryo-EM, computational and biochemical data, we investigate the oligomeric formation of HerA and detail the mechanism of nucleotide binding to the HerA-NurA complex from thermophilic archaea.

From bacterial to human dihydrouridine synthase: automated structure determination.

The reduction of uridine to dihydrouridine at specific positions in tRNA is catalysed by dihydrouridine synthase (Dus) enzymes. Increased expression of human dihydrouridine synthase 2 (hDus2) has been linked to pulmonary carcinogenesis, while its knockdown decreased cancer cell line viability, suggesting that it may serve as a valuable target for therapeutic intervention. Here, the X-ray crystal structure of a construct of hDus2 encompassing the catalytic and tRNA-recognition domains (residues 1-340) determined at 1.

Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases.

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine.

Molecular architecture of the HerA-NurA DNA double-strand break resection complex.

DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.

S-Adenosyl-S-carboxymethyl-L-homocysteine: a novel cofactor found in the putative tRNA-modifying enzyme CmoA.

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.

Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of human dihydrouridine synthase.

Dihydrouridine synthases catalyse the reduction of uridine to dihydrouridine in the D-loop and variable loop of tRNA. The human dihydrouridine synthase HsDus2L has been implicated in the development of pulmonary carcinogenesis. Here, the purification, crystallization and preliminary X-ray characterization of the HsDus2L catalytic domain are reported.

Structure and activity of a novel archaeal β-CASP protein with N-terminal KH domains.

MTH1203, a β-CASP metallo-β-lactamase family nuclease from the archaeon Methanothermobacter thermautotrophicus, was identified as a putative nuclease that might contribute to RNA processing. The crystal structure of MTH1203 reveals that, in addition to the metallo-β-lactamase nuclease and the β-CASP domains, it contains two contiguous KH domains that are unique to MTH1203 and its orthologs. RNA-binding experiments indicate that MTH1203 preferentially binds U-rich sequences with a dissociation constant in the micromolar range.

The crystal structure of unmodified tRNAPhe from Escherichia coli.

Post-transcriptional nucleoside modifications fine-tune the biophysical and biochemical properties of transfer RNA (tRNA) so that it is optimized for participation in cellular processes. Here we report the crystal structure of unmodified tRNA(Phe) from Escherichia coli at a resolution of 3 A. We show that in the absence of modifications the overall fold of the tRNA is essentially the same as that of mature tRNA.

Expression, purification, crystallization and preliminary X-ray studies of the TAN1 orthologue from Methanothermobacter thermautotrophicus.

MTH909 is the Methanothermobacter thermautotrophicus orthologue of Saccharomyces cerevisiae TAN1, which is required for N(4)-acetylcytidine formation in tRNA. The protein consists of an N-terminal near-ferredoxin-like domain and a C-terminal THUMP domain. Unlike most other proteins containing the THUMP domain, TAN1 lacks any catalytic domains and has been proposed to form a complex with a catalytic protein that is capable of making base modifications.