Ribonuclease H Enzymes Function in Bacillus subtilis

Abstract

RNA is commonly found included in chromosomal DNA forming RNA-DNA hybrids. RNA becomes embedded in DNA through DNA polymerase errors, Okazaki fragments, or annealed to DNA in the form of R-loops. Efficient processing of RNA-DNA hybrids is critical for cell survival and genome stability. RNase H enzymes are responsible for recognizing RNA-DNA hybrids and hydrolyzing the RNA containing strand. In this dissertation, I show that single ribonucleotides incorporated as DNA polymerase errors are corrected by RNase HII in a process known as ribonucleotide excision repair (RER). I show that RNase HII from B. subtilis cleaves 5' to single rNMPs embedded in DNA and that Pol I efficiently extends from an RNase HII processed substrate, reconstituting the minimal set of proteins for RER on a linear substrate in vitro. To determine the mutagenic cost that occurs in the absence of RNase HII (rnhB), mutation accumulation lines were completed demonstrating a 2-fold increase in GC → AT transitions in a strand- and sequence-context dependent manner. Using purified proteins, I demonstrate that DnaE can access a gap but not an RNase HII-dependent nick and that DnaE is ~2-fold more mutagenic than Pol I when replicating over the 3'-GCC(C/T)T-5' sequence context identified as mutagenic in vivo. This work suggests that in the absence of RNase HII a secondary pathway removes the ribonucleotide, creating a gap allowing for DnaE access and mutagenesis. To understand how RNase HII and HIII activity is regulated, I measured the activity of each RNase H enzyme on several different RNA-DNA hybrid substrates in vitro. I show that although RNase HII and HIII are capable of incising all RNA-DNA hybrids tested, the activity of RNase HII and RNase HIII is dependent on the specific divalent metal ion available to the enzyme in vitro. I demonstrate that RNase HIII from three Gram-positive bacteria are proficient for cleavage at single rNMPs embedded in DNA and that an RNase HIII nick can facilitate Pol I extension. Importantly, I show that under physiologically relevant Mg2+ and Mn2+ concentrations RNase HII efficiently cleaves RNA-DNA hybrids containing RNA-DNA junctions while RNase HIII efficiently cleaves junction-less hybrids such as R-loops. Lastly, I identify a striking sensitivity of RNase HIII deficient cells to the chemotherapeutic agent hydroxyurea (HU). Further, expression of RNase HII does not rescue the RNase HIII deficient phenotype, demonstrating different functions for these RNase H proteins in vivo. In this work, I conclude that RNase HII and Pol I are responsible for RER while RNase HIII is critical for R-loop resolution in vivo. Based on these results, I suggest that substrate specificity of RNase HII and HIII is regulated in vivo by intracellular divalent metal ion concentrations, dictating the RNA-DNA hybrids they act upon in B. subtilis.