The Impact of RNA-DNA Hybrids on Genome Integrity in Bacteria.

During the essential processes of DNA replication and transcription, RNA-DNA hybrid intermediates are formed that pose significant risks to genome integrity when left unresolved. To manage RNA-DNA hybrids, all cells rely on RNase H family enzymes that specifically cleave the RNA portion of the many different types of hybrids that form in vivo. Recent experimental advances have provided new insight into how RNA-DNA hybrids form and the consequences to genome integrity that ensue when persistent hybrids remain unresolved. Here we review the types of RNA-DNA hybrids, including R-loops, RNA primers, and ribonucleotide misincorporations, that form during DNA replication and transcription and discuss how each type of hybrid can contribute to genome instability in bacteria. Further, we discuss how bacterial RNase HI, HII, and HIII and bacterial FEN enzymes contribute to genome maintenance through the resolution of hybrids.

RnhP is a plasmid-borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610.

RNA-DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid-encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper-replication.

RecA Is Required for the Assembly of RecN into DNA Repair Complexes on the Nucleoid.

Homologous recombination requires the coordinated effort of several proteins to complete break resection, homologous pairing, and resolution of DNA crossover structures. RecN is a conserved bacterial protein important for double-strand break repair and is a member of the structural maintenance of chromosomes (SMC) protein family. Current models in Bacillus subtilis propose that RecN responds to double-stranded breaks prior to RecA and end processing, suggesting that RecN is among the very first proteins responsible for break detection. Here, we investigate the contribution of RecA and end processing by AddAB to RecN recruitment into repair foci in vivo. Using this approach, we found that recA is required for RecN-green fluorescent protein (GFP) focus formation on the nucleoid during normal growth and in response to DNA damage. In the absence of recA function, RecN foci form in a low percentage of cells, RecN localizes away from the nucleoid, and RecN fails to assemble in response to DNA damage. In contrast, we show that the response of RecA-GFP foci to DNA damage is unchanged in the presence or absence of recN. In further support of RecA activity preceding RecN, we show that ablation of the double-strand break end-processing enzyme addAB results in a failure of RecN to form foci in response to DNA damage. With these results, we conclude that RecA and end processing function prior to RecN, establishing a critical step for the recruitment and participation of RecN during DNA break repair in Bacillus subtilis. IMPORTANCE Homologous recombination is important for the repair of DNA double-strand breaks. RecN is a highly conserved protein that has been shown to be important for sister chromatid cohesion and for surviving break-inducing clastogens. Here, we show that the assembly of RecN into repair foci on the bacterial nucleoid requires the end-processing enzyme AddAB and the recombinase RecA. In the absence of either recA or end-processing RecN-GFP, foci are no longer DNA damage inducible, and foci form in a subset of cells as large complexes in regions away from the nucleoid. Our results establish the stepwise order of action, where double-strand break end processing and RecA association precede the participation of RecN in break repair in Bacillus subtilis.