DNA replication and transcription machineries function simultaneously, on the same template, leading to conflicts between the two. During conflicts, the nascent mRNAs can hybridize to their template strands, generating RNA:DNA hybrids, which reside within nucleic acid structures known as R-loops. Accumulation of R-loops can lead to severe replication fork stalling, eventually leading to cell death. In bacteria, there are well-known replication restart mechanisms that should rescue these stalled replication forks, and yet, during conflicts, this process is inhibited. Additionally, in vitro work has shown that RNA:DNA hybrids are substrates for replication restart. Here, we discovered that the highly conserved exonuclease Polynucleotide Phosphorylase (PNPase in Bacillus subtilis) prevents replication restart from RNA:DNA hybrids, specifically at regions of severe conflicts where R-loops accumulate. Our in vitro data show that PNPase binds RNA:DNA hybrids and digests the RNA in these structures. Consistently, we found that in vivo, PNPase binds to conflict regions and reduces R-loop levels. To our knowledge, the only other class of enzymes known to digest the RNA from hybrids are RNases H. Our findings identify PNPase as a new enzyme that can perform a similar function. Importantly, our data show that PNPase inhibits replication restart from R-loops and its absence allows for replication restart from conflict regions. We also observed that PNPase activity reduces mutations, suggesting that replication restart from RNA:DNA hybrids is highly mutagenic. We propose that PNPase acts as a safeguard against mutagenic replication restart from RNA:DNA hybrids within R-loops by digesting the RNA which could re-initiate replication.
Sensoy, O., Carvajal-Garcia, J., Boumi, S., Merrikh, H.
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