RNase H2 roles in genome integrity revealed by unlinking its activities

Abstract

Ribonuclease H2 (RNase H2) protects genome integrity by its dual roles of resolving transcription-related R-loops and ribonucleotides incorporated in DNA during replication. To unlink these two functions, we generated a Saccharomyces cerevisiae RNase H2 mutant that can resolve R-loops but cannot cleave single ribonucleotides in DNA. This mutant definitively correlates the 2-5 bp deletions observed in rnh201Δ strains with single rNMPs in DNA. It also establishes a connection between R-loops and Sgs1-mediated replication reinitiation at stalled forks and identifies R-loops uniquely processed by RNase H2. In mouse, deletion of any of the genes coding for RNase H2 results in embryonic lethality, and in humans, RNase H2 hypomorphic mutations cause Aicardi-Goutières syndrome (AGS), a neuroinflammatory disorder. To determine the contribution of R-loops and rNMP in DNA to the defects observed in AGS, we characterized in yeast an AGS-related mutation, which is impaired in processing both substrates, but has sufficient R-loop degradation activity to complement the defects of rnh201Δ sgs1Δ strains. However, this AGS-related mutation accumulates 2-5 bp deletions at a very similar rate as the deletion strain.

Figure 1.

Comparison of RNase H2 and H3. (A) Alignment was generated for human RNase H2A (Hu2A), S. cerevisiae RNase H2A (Sc2A), T. maritima RNase H2 (Tm2), B. stearothermophilus RNase H3 (Bst3), Streptococcus pneumonia RNase H3 (Spn3) and Thermovibrio ammonificans HB-1 RNase H3 (Tam3). α-Helices are indicated with pink letters, and β-sheets are indicated with orange letters. Active site residues are highlighted in yellow. Amino acid substitutions in S. cerevisiae RNase H2A are noted above the first row of alignment D39A, G42S, P45D, S or E and Y219A—along with the conserved DSK triplet. The ‘GRG’ motif, DSK and the conserved Tyr of RNase H2 are also highlighted in grey. (B) The 5′-³²P-labeled 12 mer substrates indicated above the gels were digested by yeast and Tm-RNase H2 in the presence of 10 mM MgCl₂. The lanes marked with 0 contained no enzyme, and lanes marked with triangle contained increasing amount of the proteins (0.16, 1.6, 16 and 160 nM in the case of Tm-RNase H2, 0.011, 0.11, 1.1 and 11 nM in the case of yeast RNase H2 and 0.011, 0.11, 1.1 and 11 nM in the case of Bst-RNase H3). Products of the hydrolysis were analyzed by 20% TBE-urea gels. The sizes of products were measured based on molecular size markers indicated as M (products of digestion of ³²P-labeled strands without complementary strand by phosphodiesterase I). Major cleavage sites of the substrates are summarized on the bottom of each gel. (C) Relative activity of Bst-RNase H3 D103N and D103A mutants compared with the wild-type protein was analyzed by liquid RNase H assay using poly-rA/poly-dT substrate as previously done (15).

Figure 2.

Models of Bst-RNase H3 and Tm-RNase H2 in complex with substrates. The ribbon models of Bst-RNase H3 (in green) and Tm-RNase H2 (in orange) are superimposed, and the complexes with (A) duplex DNA containing single ribonucleotide (DNA in blue and RNA in red) and (B) RNA/DNA hybrid are shown. Active site residues (DEDD motif in Tm-RNase H2 and DEDE motif in Bst-RNase H3) and residues involved in recognition of (C) DpRpD and (D) RpRpR substrates (GRG/Y motif in Tm-RNase H2, GTGD motif in Bst-RNase H3) are shown. Possible hydrogen bonds are indicated by dotted lines. The nucleotides are numbered relative to the scissile phosphate, which is indicated with an arrow. Four catalytic residues (DEDD for Tm-RNase H2 and DEDE for Bst-RNase H3) are also indicated in numerical order.

Figure 3.

Cleavage of poly-rA/poly-dT substrate and oligo substrates with Sc-RNase H2 WT and mutants. (A) The uniformly ³²P-labeled poly-rA/poly-dT substrate (1 µM) was digested by the Sc-RNase H2 WT and mutants indicated above the gel in the presence of 10 mM MgCl₂. The lanes marked with 0 contained no enzyme, and lanes marked with triangle contained increasing amount of the proteins (1 pM, 11 pM, 110 pM, 1.1 nM and 11 nM). Products of the hydrolysis were analyzed by 12% TBE-urea gels and visualized by Phosphorimager. (B) The 5′-³²P-labeled 12 mer substrates indicated on the left of the gels were digested by Sc-RNase H2 in the presence of 10 mM MgCl₂. The lanes marked with 0 contained no enzyme, and lanes marked with triangle contained increasing amount of the proteins (0.011, 0.11, 1.1 and 11 nM). Products of the hydrolysis were analyzed by 20% TBE-urea gels. The sizes of products were measured based on molecular size markers indicated as M. Cleavage at the 5′ of RpD junction is indicated by an arrow.

Figure 4.

In vitro RER assay. (A) In vitro RER assay is schematically shown. The 7.3-kb mp18 single-stranded circle DNA was replicated by Polδ and cofactors in the presence of the physiological levels of dNTPs, rNTPs and ³²P-labeled dATP at 30°C. After 12 min incubation, FEN1, Ligase1 and wild-type or mutant RNase H2 were added, and the reactions were continued. After 1 or 3 min, aliquots were taken and treated with NaOH for hydrolysis at RpD linkage due to misincorporation of ribonucleotide during replication. DNA was extracted from the aliquots and loaded onto 1% alkali agarose gel. The alkali agarose gel was visualized by Phosphorimager. (B) The radioactivity distribution was scanned and divided by the size distribution to obtain a normalized product distribution as described before (39).

Figure 5.

In vivo phenotypes of strains rnh201Δ carrying plasmids harboring RNH201 WT and mutants. (A) Saccharomyces cerevisiae strain pol2-M644G rnh201Δ was transformed with plasmid expressing RNH201WT and mutants and empty vector. Strains were plated in canavanine plates and the rates of individual mutations types at CAN1 determined. N, number of mutants sequenced; indel, insertion/deletion/; BS, base substitution. 95% confidence intervals are in parenthesis below total rates. (B) Saccharomyces cerevisiae strain YAEH275 (BY4741 but P_GAL1-3HA-TOP1 (KanMx6) rnh201Δ (NatMx6) rnh1Δ (HphMx6)) was transformed with plasmids expressing RNH201-WT, rnh201-G42S, -P45D-Y219A and -D39A and also an empty vector. The transformants were grown at 30°C in liquid SD-Leu medium containing 2% galactose, and series of dilutions were spotted on SD-Leu plates with 2% galactose or 2% glucose and incubated for 3 days at 30°C.

Figure 6.

Effect of RNase H2-mutations in sgs1Δ yeast strain. (A) Doubling time of the yeast strains with RNase H2-mutations in genomic DNA was determined at 30°C in YPD medium. Error bars are standard deviations with P < 0.1 measured by the Ttest in Excel. (B) Representatives of normal cells and abnormal cells from sgs1Δ rnh201Δ background stained with DAPI are shown. (C) Fraction of abnormal cells from log-phase culture was analyzed by gating enlarged cells by FACS, which was correlated with the ratio of abnormal cells observed by microscope (data not shown).

Figure 7.

Effects of RNase H2 mutations in single ribonucleotide processing and R-loop resolution. (A) Single ribonucleotides incorporate in the genome can be processed by RNase H2. RNase H2-P45D-Y219A mutant cannot remove single rNMPs and the AGS-related RNase H2-G42S has low but detectable activity. Both mutations lead to the accumulation of 2–5 bp deletions in genomic DNA, although RNase H2-G42S at a lower rate. (B) R-loops that form during transcription are processed primarily by RNase H1. RNase H2 WT and P45D-Y219A mutant can process equally well these structures, whereas RNase H2-G42S is not as effective. (C) R-loops involved in replication fork collapse have unique access to RNase H2. The WT enzyme and the mutants, RNase H2-G42S and RNase H2-P45D-Y219A, are equally active on these substrates.