RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• R-loops, formed by RNA:DNA hybrids, are a significant source of replication stress.
• The precise mechanisms by which R-loops impede DNA replication fork progression are not fully understood.

Purpose of the Study:

• To investigate how RNA:DNA hybrids affect DNA replication fork progression, arrest, and restart.
• To elucidate the role of RNase H enzymes in resolving R-loops and maintaining replication fidelity.

Main Methods:

• Utilized Saccharomyces cerevisiae and human cell lines lacking RNase H1 and/or H2.
• Monitored replication fork dynamics under challenged (hydroxyurea, methyl methanesulfonate) and unchallenged conditions.
• Assessed RNA:DNA hybrid levels, single-stranded DNA (ssDNA) formation, and nascent DNA resection at stalled forks.
• Investigated the effect of triptolide, a transcription inhibitor, on fork repair.

Main Results:

• RNase H-deficient cells exhibited impaired replication and increased RNA:DNA hybrids at stalled forks under stress.
• These cells failed to generate RPA-coated ssDNA, a critical intermediate for replication restart.
• Similar defects in nascent DNA resection and ssDNA formation were observed in human RNase H2-deficient cells.
• Triptolide treatment restored fork resection in human cells, suggesting transcription interference.

Conclusions:

• RNA:DNA hybrids act as physical barriers to replication forks.
• Beyond blocking progression, R-loops interfere with essential post-replicative fork repair mechanisms.
• Efficient degradation of RNA:DNA hybrids by RNase H is vital for preventing replication stress and ensuring genome stability.