Substrate Specificity for Bacterial RNases HII and HIII Is Influenced by Metal Availability

Abstract

We tested the activities of four predicated RNase H enzymes, including two RNase HI-type enzymes, in addition to RNase HII (RnhB) and RNase HIII (RnhC), on several RNA-DNA hybrid substrates with different divalent metal cations. We found that the two RNase HI-type enzymes, YpdQ and YpeP, failed to show activity on the three substrates tested. RNase HII and RNase HIII cleaved all the substrates tested, although the activity was dependent on the metal made available. We show that Bacillus subtilis RNase HII and RNase HIII are both able to incise 5' to a single ribonucleoside monophosphate (rNMP). We show that RNase HIII incision at a single rNMP occurs most efficiently with Mn²⁺, an activity we found to be conserved among other Gram-positive RNase HIII enzymes. Characterization of RNases HII and HIII with metal concentrations in the physiological range showed that RNase HII can cleave at single rNMPs embedded in DNA while RNase HIII is far less effective. Further, using metal concentrations within the physiological range, RNase HIII efficiently cleaved longer RNA-DNA hybrids lacking an RNA-DNA junction, while RNase HII was much less effective. Phenotypic analysis showed that cells with an rnhC deletion were sensitive to hydroxyurea (HU). In contrast, cells with an rnhB deletion showed wild-type growth in the presence of HU, supporting the hypothesis that RNases HII and HIII have distinct substrate specificities in vivo This work demonstrates how metal availability influences the substrate recognition and activity of RNases HII and HIII, providing insight into their functions in vivoIMPORTANCE RNase H represents a class of proteins that cleave RNA-DNA hybrids, helping resolve R-loops and Okazaki fragments, as well as initiating the process of ribonucleotide excision repair (RER). We investigated the activities of four Bacillus subtilis RNase H enzymes and found that only RNases HII and HIII have activity and that their substrate preference is dependent on metal availability. To understand the factors that contribute to RNase HII and RNase HIII substrate preference, we show that in the presence of metal concentrations within the physiological range, RNases HII and HIII have distinct activities on different RNA-DNA hybrids. This work provides insight into how RNases HII and HIII repair the broad range of RNA-DNA hybrids that form in Gram-positive bacteria.

Keywords: Bacillus subtilis; DNA repair; R-loop; RNA-DNA hybrid; RNase; ribonucleotide excision repair.

FIG 1

B. subtilis has two active RNase H enzymes. (A) Alignment of two putative RNase H enzymes, YpdQ and YpeP, with E. coli RNase HI. B. subtilis RNases HII and HIII aligned with E. coli RNase HII. Conserved residues are shaded in gray, catalytic residues are in boldface, and residues involved in substrate binding have a line above. The alignment was performed using Clustal Omega. Asterisks denote the GRG motif. (B) SDS-PAGE with 2 μg of the indicated B. subtilis RNase H enzymes stained with Coomassie blue. (C) Four nanomolar RNase HII and HIII (left) and 0.5 μM YpeP and YpdQ (right) cleavage of a 5′-end-labeled 20-bp RNA-DNA hybrid with Mg²⁺, Mn²⁺, or Co²⁺ resolved by 20% urea-PAGE. (D) Four nanomolar RNase HII and HIII (left) and 0.5 μM YpeP and YpdQ (right) cleavage of a 5′-end-labeled four-embedded-rNMP hybrid substrate with Mg²⁺, Mn²⁺, or Co²⁺ resolved by 20% urea-PAGE. (E) Fifty nanomolar RNases HII and HIII (left) and 0.5 μM YpeP and YpdQ (right) incubated with a 5′-end-labeled single-embedded-rNMP RNA-DNA hybrid with Mg²⁺, Mn²⁺, or Co²⁺ resolved by 20% urea-PAGE. (C to E) Metal concentrations were 2 mM. A red bar or “r” indicates RNA; an asterisk indicates the position of the 5′ label for the substrate.

FIG 2

Cleavage of a single embedded rNMP with Mn²⁺ is conserved among different RNase HIII enzymes. (A) A 5′-end-labeled single-embedded-rNMP substrate cleaved with 25, 50, and 100 nM RNase HII and RNase HIII with Mg²⁺ (top) or Mn²⁺ (bottom). DE/AA corresponds to 1 μM RNase HII D78A and E79A and HIII D100A and E101A included as controls. Metal concentrations were 1 mM. (B) Predicted functional domains of B. subtilis (Bsu) RNase HIII. An alignment of the region containing the G(R/K)G motif from C. pneumoniae (Cpn) (boldface) is shown magnified below. C. pneumoniae S94 is in blue. Gst, G. stearothermophilus; Sau, S. aureus. (C) Phylogenetic tree of some RNase HIII-containing bacteria based on N-terminal domain (NTD) homology. Blue, RNase HIII assayed in panel D; boldface, RNase HIII published previously (26, 28). (D) Cleavage of a 5′-end-labeled four-embedded-rNMP (top) or single-embedded-rNMP (bottom) hybrid substrate with RNase HIII from B. subtilis (wild type) D100A and E101A, G. stearothermophilus, and S. aureus with Mg²⁺ (left) or Mn²⁺ (right) using 1 mM metal concentrations. “r” represents the number and position of ribonucleotides; the asterisk indicates the position of the 5′ label.

FIG 3

RNase HII is effective on substrates with an RNA-DNA junction using metal concentrations in the physiological range. (A) Four nanomolar RNase HII cleaving a 20-bp RNA-DNA hybrid substrate with varying Mg²⁺ and Mn²⁺ concentrations. (B) Four nanomolar RNase HII cleaving a substrate with 4 embedded rNMPs in DNA with varying concentrations of Mg²⁺ and Mn²⁺. (C) Fifty nanomolar RNase HII cleaving a single embedded rNMP in DNA, representing an RER substrate, with varying Mg²⁺ and Mn²⁺ concentrations. (A to C) Predicted in vivo concentrations (29) of Mg²⁺ and Mn²⁺ are shown in boldface. The percentage of substrate cleaved versus the metal concentration is graphed below. The concentrations of Mg²⁺ and Mn²⁺ used were in 10-fold increments from 0.001 mM to 10 mM. The mean is reported, with error bars representing the range of duplicate samples. For the substrate, the red bar or “r” indicates RNA. The asterisk indicates the 5′ label.

FIG 4

RNase HIII is effective on a substrate lacking an RNA-DNA junction using metal concentrations in the physiological range. (A) Four nanomolar RNase HIII cleaving a 20-bp RNA-DNA hybrid substrate with varying Mg²⁺ and Mn²⁺ concentrations. (B) Four nanomolar RNase HIII cleaving a substrate with 4 embedded rNMPs in DNA with varying concentrations of Mg²⁺ and Mn²⁺. (C) Fifty nanomolar RNase HIII cleaving a single embedded rNMP in DNA, representing an RER substrate, with varying Mg²⁺ and Mn²⁺ concentrations. (A to C) Predicted in vivo concentrations (29) of Mg²⁺ and Mn²⁺ are in boldface. The percentage of substrate cleaved versus the metal concentration is graphed below. The concentrations of Mg²⁺ and Mn²⁺ used were in 10-fold increments from 0.001 mM to 10 mM. The mean is reported, with error bars representing the range of duplicate samples. The red bar or “r” indicates RNA. The asterisk indicates the 5′ label.

FIG 5

DNA polymerase I can extend from an RNase HIII-incised substrate. (A) Schematic of the experimental design with a single embedded rNMP in duplex DNA cleaved by RNase H or hydrolyzed by NaOH, followed by extension with Pol I. The red bar indicates a single ribonucleotide. (B) A single embedded rNMP substrate with no cleavage (None), NaOH hydrolysis (NaOH), RNase HII cleavage (RNase HII), or RNase HIII cleavage (RNase HIII), followed by incubation with Pol I for 5, 25, and 75 min. For efficient incision, RNases HII and HIII were incubated with 2 mM MnCl₂. (C) Pol I-incubated products from panel B were treated with NaOH for 60 min to determine if the product following Pol I extension was sensitive or refractory to alkaline treatment to test for removal and replacement of the rNMP with a dNMP.

FIG 6

Cells lacking RNase HIII are sensitive to hydroxyurea (HU). (A) Spot titer assay of isogenic B. subtilis cells with the indicated genotype spotted on HU. (B) Spot titer assay of isogenic B. subtilis cells with the indicated genotype spotted on HU. IPTG was included at 1 mM. (C) Immunoblot detection of RNase HII from whole-cell extracts. Detection was accomplished using affinity-purified antiserum raised against RNase HII (RnhB) as the primary antibody.