Evidence for DNA cleavage caused directly by a transfer RNA-targeting toxin

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The killer yeast Pichia acaciae produces a protein complex, PaT, that induces DNA damage and cell cycle arrest in Saccharomyces cerevisiae.
• A subunit of PaT, Orf2p, was previously shown to cleave a specific transfer RNA (tRNA), but the mechanism of DNA damage remained unclear.

Purpose of the Study:

• To elucidate the mechanism by which Orf2p induces DNA damage and cell cycle arrest in Saccharomyces cerevisiae.
• To determine if Orf2p directly cleaves genomic DNA or if the damage is solely a consequence of tRNA cleavage.

Main Methods:

• In vitro assays to assess the enzymatic activity of Orf2p on both tRNA and genomic DNA.
• Site-directed mutagenesis of the His299 residue in Orf2p to investigate its role in DNA and tRNA cleavage.
• Analysis of cell cycle progression and histone phosphorylation in yeast cells expressing wild-type or mutant Orf2p.

Main Results:

• Orf2p was demonstrated to cleave genomic DNA in vitro, in addition to its previously identified tRNA cleavage activity.
• The catalytic activity of Orf2p on both DNA and tRNA requires the same histidine residue (His299).
• Substitution of His299 with alanine abolished both DNA/tRNA cleavage and the resulting cell cycle arrest, indicating His299 is essential for toxicity.

Conclusions:

• The DNA damage induced by Orf2p is a direct consequence of its enzymatic activity on genomic DNA, not solely due to tRNA cleavage.
• Impairment of translation via tRNA cleavage allows Orf2p nuclear entry, leading to histone phosphorylation and cell cycle arrest.
• Orf2p represents a novel class of DNA-damaging toxins with potential implications for understanding yeast genetics and developing new antifungal strategies.