Single-molecule dynamics of ribonuclease H enzymes in Bacillus subtilis.

Abstract

Transcription and replication are fundamental processes for gene expression during which RNA-DNA hybrids (such as R-loops, single-ribonucleotide errors, RNA primers, ribopatches, and complementary RNA:DNA) may form. When RNA-DNA hybrids are not resolved, replication fork stress can occur leading to compromised genome integrity and an increase in cell mutation rates. Ribonuclease (RNase) H family enzymes contribute to genome maintenance by cleaving the RNA portion of RNA-DNA hybrid structures. Recent work has deepened our understanding of how RNA-DNA hybrids form and what could occur if they are not resolved. However, how these enzymes are recruited to the sites of RNA-DNA hybrids and the individual contributions of each enzyme remain unclear. Here, we use live-cell single-molecule imaging and super-resolution microscopy techniques to study the dynamics of the two RNase H enzymes in Bacillus subtilis. We observe changes in the single-molecule dynamics and localization patterns of each RNase H enzyme upon arresting transcription and inhibiting replication. Our results show that inhibition of transcription and replication both affect the fraction of enzymes existing in a functional binding state, as well as the localization of the enzymes at the replication fork. Our work provides insight into the specific functions of RNase H enzymes for different types of RNA-DNA hybrids in B. subtilis.