Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Genome instability can result from RNA:DNA hybrids (R-loops) and ribonucleotide monophosphate (rNMP) incorporation into DNA.
• Ribonuclease (RNase) H1 and RNase H2 are key enzymes involved in resolving these structures.
• RNase H1 and RNase H2 degrade RNA in R-loops, while RNase H2 also removes rNMPs from DNA.

Purpose of the Study:

• To investigate the distinct contributions of R-loops and rNMPs to genome instability.
• To determine the role of RNase H1 and RNase H2 in maintaining genome stability through high-resolution mapping of recombination events.
• To elucidate the primary cause of elevated recombination in RNase H2-deficient yeast strains.

Main Methods:

• High-resolution mapping of mitotic recombination events in Saccharomyces cerevisiae.
• Comparative analysis of genome-wide recombination in wild-type, rnh1Δ, rnh201Δ, and rnh1Δ rnh201Δ yeast strains.
• Assessment of recombination levels in an rnh201Δ mutant with a reduced rNMP incorporation mutation (pol2-M644L).

Main Results:

• Deletion of RNase H1 (rnh1Δ) had minimal impact on recombination rates.
• Significant elevation in recombination was observed in both rnh201Δ and double-mutant (rnh1Δ rnh201Δ) strains.
• Recombination levels in the double mutant were approximately 50% higher than in the rnh201 single mutant.
• An rnh201Δ mutant with reduced rNMP incorporation showed similar instability to wild-type strains, suggesting R-loops are the main driver.

Conclusions:

• RNase H2 plays a critical role in preventing genome instability, primarily by resolving R-loops.
• The elevated recombination observed in RNase H2-deficient cells is predominantly caused by R-loops, rather than rNMP incorporation.
• Understanding the distinct roles of RNase H1 and H2 is crucial for comprehending genome stability mechanisms.