DDX37 and DDX50 Maintain Genome Stability by Preventing Transcription-dependent R-loop Formation.

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• R-loops, structures of RNA-DNA hybrids, are vital in biological processes but can threaten genome stability if aberrant.
• Strict control of R-loop formation and resolution is essential, involving RNAse H and helicases.

Purpose of the Study:

• To identify novel RNA helicases involved in maintaining genome integrity by interacting with RNA-DNA hybrids.
• To elucidate the function of DDX37 and DDX50 in R-loop resolution and prevention of DNA damage.

Main Methods:

• Screening for RNA helicases interacting with RNA-DNA hybrids.
• Depletion of DDX37 and DDX50 using knockdown techniques.
• Assessing DNA damage via H2AX phosphorylation and comet assays.
• Evaluating replication stress through DNA replication track length and RPA focus formation.
• Measuring RNA-DNA hybrid levels and assessing the effect of RNase H1 overexpression and transcription inhibition.

Main Results:

• DDX37 and DDX50 were identified as novel RNA helicases controlling genome integrity.
• Depletion of DDX37/DDX50 led to increased DNA damage (H2AX phosphorylation, comet tails) and replication stress (reduced replication track length, RPA foci).
• Knockdown of DDX37/DDX50 increased RNA-DNA hybrids, which was reversible by RNase H1, and prevented by inhibiting transcription, indicating their role in resolving transcription-associated R-loops.

Conclusions:

• DDX37 and DDX50 are essential for resolving RNA-DNA hybrids that arise during transcription.
• These helicases play a critical role in preventing DNA damage induced by replication stress stemming from aberrant R-loops.