Histone Mutants Separate R Loop Formation from Genome Instability Induction

Area of Science:

• Molecular Biology
• Genetics
• Epigenetics

Background:

• R loops, structures of DNA-RNA hybrids, have essential physiological functions.
• However, R loops can also lead to genome instability, but the underlying mechanisms remain unclear.

Purpose of the Study:

• To investigate the mechanisms by which R loops cause genome instability.
• To identify genetic factors that influence R loop formation and their impact on genomic integrity.

Main Methods:

• Identification of yeast histone H3 and H4 mutations that facilitate R loops without causing instability.
• Utilizing ssDNA-specific human AID and bisulfite to characterize R loops, distinguishing between RNA-DNA hybrids and ssDNA-containing R loops.
• Analyzing H3 serine-10 phosphate (H3S10-P) levels in different mutant strains.

Main Results:

• Specific histone mutations were found to facilitate R loops but did not induce genome instability.
• R loops containing single-stranded DNA (ssDNA) were detected and were similar in size regardless of instability induction.
• Mutants facilitating R loops without instability did not accumulate H3S10-P, unlike mutants causing instability.
• These histone mutations suppressed the elevated H3S10 phosphorylation and genomic instability observed in hpr1 and sen1 mutants.

Conclusions:

• R loop-induced genome instability is a two-step process: initial R loop formation facilitated by altered chromatin, followed by chromatin modification (including H3S10-P) that compromises integrity.
• R loops themselves are not sufficient to cause genome instability; subsequent chromatin modifications are required.
• Histone modifications play a critical role in regulating the potentially deleterious effects of R loops on genome stability.