Characterization of six recombinant human RNase H2 bearing Aicardi-Goutiéres syndrome causing mutations

Abstract

Mammalian RNase H2 is a heterotrimeric enzyme consisting of one catalytic subunit (A) and two accessory subunits (B and C). RNase H2 is involved in the removal of a single ribonucleotide embedded in genomic DNA and removal of RNA of RNA/DNA hybrids. In humans, mutation of the RNase H2 gene causes a severe neuroinflammatory disorder Aicardi-Goutières syndrome (AGS). Here, we examined the activity and stability of six recombinant human RNase H2 variants bearing one AGS-causing mutation, A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G, or C-R69W. The activity of A-G37S was 0.3–1% of that of the wild-type RNase H2 (WT), while those of other five variants were 51–120%. In circular dichroism measurement, the melting temperatures of variants were 50–53°C, lower than that of WT (56°C). These results suggested that A-G37S had decreased activity and stability than WT, while other five variants had decreased stability but retained activity. In gel filtration chromatography of the purified enzyme preparation, WT migrated as a heterotrimer, while A-R291H eluted in two separate peaks containing either the heterotrimer or only the A subunit, suggesting that some AGS-causing mutations affect the heterotrimer-forming stability of RNase H2.

Ribonuclease H (RNase H) specifically hydrolyzes the 5’-phosphodiester bond of the RNA of RNA/DNA hybrids. RNase H is classified into two groups based of tertiary structure. Type I in prokaryotes and eukaryotes has RNA strand degrading activity but no single ribonucleotide (rNMP) incision activity. In prokaryotes, type II RNases H fall into two classes: (i) RNase HII exhibit cleavage mainly at rNMP site in DNA and (ii) RNase HIII which only cleaves RNA/DNA (1–3). Eukaryotic RNases H2 are able to incise at single rNMPs in DNA and hydrolyzes RNA of RNA/DNA. Eukaryotic type 2 RNase H (RNase H2) is a heterotrimer consisting of one catalytic subunit (A) and two accessory subunits (B and C) (4–6).

Each of the three replicative DNA polymerases incorporate a single ribonucleotide every few thousand base pairs, which if remain unrepaired such ribonucleotides can cause double strand DNA breaks, leading to mutation, cell death and onset of cancer and other genetic diseases (7–9). RNase H2 is involved in the removal of ribonucleotides embedded in genomic DNA, via the process of ribonucleotide excision repair (7). Loss of Rnaseh2A (10), 2B or 2 C (11, 12) in mice leads to early embryonic lethality. Recent results have shown that RNase H2 A-subunit gene knockout leads to the accumulation of ribonucleotides in genomic DNA in NIH3T3 cells (13) and abolishes retroelement propagation in HEK293T cells (14). The yeast RNase H2 mutant bearing a double mutation P45D/Y219A in the A subunit only has RNA strand degrading activity and lacks a single ribonucleotide excision activity (15). This mutant was termed a ribonucleotide excision defective (RED) variant (15). RNase H2 A-subunit gene knockout Rnaseh2a^–/– and Rnaseh2a^RED/RED mice are both embryonic lethal (10), indicating lethality due to abundant ribonucleotides in DNA as cause of death.

In Aicardi-Goutières syndrome (AGS), a severe neuroinflammatory disorder (16–20), human patients have bi-allelic mutations in any of seven genes (RNASEH2A, RNASEH2B, RNASEH2C, TREX1, SAMHD1, ADAR, or IFIH1). Mutations affecting RNase H2 account for more than 50% of the total AGS patients. The majority of the RNase H2 defects are found in the RNASEH2B gene, accounting for 36% of the total AGS patients as of 2015 (20). Mutations in RNASEH2A (5%) and RNASEH2C (12%) are less frequently found (20).

To explore the effects of AGS-causing mutations on AGS, it is important to analyse in detail how AGS-causing mutations affect the activity and stability of RNase H2. We previously examined the characteristics of the recombinant wild-type (WT) human RNase H2 expressed in Escherichia coli (21). In this study, we prepared six recombinant human RNase H2 variants bearing the mutation in G37S, N212I, or R291H in the A subunit, A177T or V185G in the B subunit, or R69W in the C subunit and characterized the activities including the salt dependence and the stabilities.

Materials and Methods

Expression and purification of RNase H2 variants

For the expression of the WT human RNase H2, E. coli BL21(DE3) [F^–, ompT, hsdS_B(r_B^–m_B^–) gal dcm (DE3)] was used as a host. pET15b-hH2ABC, which was the pET-15b(+) plasmid (Merck Bioscience, Tokyo, Japan) harbouring the gene encoding A, B and C subunits of human RNase H2 with a N-terminal (His)₆ tag at each subunit, was used as the expression plasmid of WT. Expression plasmids of variants were constructed by site-directed mutagenesis using the pET15b-hH2ABC as a template and the oligonucleotides listed in Supplementary Table SI. The nucleotide sequences of mutated RNase H2 genes were verified.

WT and variants were prepared as described previously (21). Briefly, the overnight culture of the BL21(DE3) transformants (10 ml) was added to 1000 ml of LB broth containing 50 μg/ml ampicillin in a 2-liter flask and incubated at 30°C under vigorous aeration by air-pump. When OD₆₆₀ reached 0.3, 1 ml of 0.5 M IPTG was added and growth was continued at 30°C for 3 h. After centrifugation at 10,000 × g for 10 min, the cells were harvested, suspended with 20 ml of 20 mM Tris-HCl buffer (pH 7.5), 1 mM EDTA, 1 mM dithiothreitol (buffer A) and disrupted by sonication. After centrifugation at 20,000 × g for 40 min, the supernatant was collected and applied to a HiTrap Heparin HP column (GE Healthcare, Buckinghamshire, UK) pre-equilibrated with buffer A. After the wash with buffer A containing 0.2 M NaCl, the bound RNase H2 was eluted with buffer A containing 0.4 M NaCl and applied to a HisTrap HP column (GE Healthcare) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5), 0.5 M NaCl (buffer B). After the wash with buffer B, the bound RNase H2 was eluted with buffer B containing 30 mM imidazole and applied to a PD-10 column (GE Healthcare) pre-equilibrated with Tris-HCl buffer (pH 8.3), 200 mM KCl, 50% glycerol. Purified enzyme solution was stored at −80°C before use. The enzyme concentration was determined using the molar absorption coefficient at 280 nm of 83,030 M⁻¹ cm⁻¹.

Circular dichroism measurement

A Jasco J-820 (Tokyo, Japan) spectropolarimeter equipped with a Peltier system of cell temperature control was used. The spectrometer conditions were: spectral range 210–340 nm; 100 mdeg sensitivity; 0.1 nm resolutions; 4 s response time; 20 nm min⁻¹ scan rate and 5 accumulations. The control baseline was obtained with solvent and all the components without RNase H2. Circular dichroism (CD) spectra were recorded at 25°C using 2-mm cell. The concentration of RNase H2 was 1.0 μM in 5 mM Tris-HCl buffer (pH 8.3), 20 mM KCl, 5% glycerol. CD spectra were processed with a Jasco software and finally expressed in mean-residue molar ellipticity units, [θ] (deg cm² dmol⁻¹).

For the analysis of thermal denaturation of RNase H2, the solution (500 μl) containing RNase H2 (1.0 μM) in 5 mM Tris-HCl buffer (pH 8.3), 20 mM KCl, 5% glycerol was incubated at 25°C for 5 min. After the incubation, the solution (400 μl) was transferred to a 2-mm cell, and mineral oil (50 μl) was added to avoid evaporation. Thermal denaturation was examined by monitoring the CD value at 222 nm, θ₂₂₂, from 30 to 70°C at 1°C/min.

RNase H2 assay

RNase H2 assay was performed using a fluorescence substrate as described previously (21, 22). Briefly, an RNA₁₈/DNA₁₈ hybrid (named R18/D18) was prepared by incubating 1.0 μl of 100 μM 3’-FITC labelled 18-mer RNA 5’-gaucugagccugggagcu-FITC-3’ (R18) (Fasmac, Atsugi, Japan) and 1.2 μl of 100 μM 5’-Dabcyl labelled 18-mer DNA 5’-Dabcyl-AGCTCCCAGGCTCAGATC-3′ (D18) (Fasmac) in 50 mM Tris-HCl buffer (pH 8.0) containing 60 mM KCl at 25°C for 30 min. A hybrid consisting of DNA₁₄-RNA₁-DNA₃ and DNA₁₈ (named R1/D18) was prepared as described above by using 3’-FITC labelled 5’-GATCTGAGCCTGGGaGCT-FITC-3’ (R1) instead of R18. Enzyme reaction was started by adding 20 μl of RNase H2 solution (0.5–5 nM for WT and variants except for A-G37S and 25–200 nM for A-G37S) to the 180 μl of 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl₂, 60 mM KCl, either of 5.6 nM R1/D18 or 5.6 nM R18/D18 in a 96-well plate. The reaction was monitored by following the increase in fluorescence intensity at 515 nm with excitation at 490 nm with an EnSight (PerkinElmer, Waltham, MA) every 5 s for 5 min.

Gel filtration chromatography

Human RNase H2 [0.1 ml of 1.7 μM in 20 mM Tris-HCl buffer (pH 7.5)] was applied onto a COSMOSIL Packed Column 5Diol–300–II (7.5 mm inner diameter x 600 mm) (Nacalai Tesque, Kyoto, Japan) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5), 0.5 M l-Arg at a flow-rate of 1.0 ml/min and detected by absorbance at 280 nm (A₂₈₀).

Results and Discussion

Production of recombinant human RNase H2 variants

Figure 1 shows the structure of human RNase H2 with the amino acid residues to be mutated (6, 23). The C subunit is flanked by the A and B subunits. The C-terminal region of the A subunit interacts with the C and B subunits and emerges from the B subunit becoming visible near the top of the structure. Gly37 and Asn212 in the A subunit are located close to the active site and distant from the B and C subunits. Arg291 is located in the C-terminus of the A subunit, distant from the active site and interacting with the B and C subunits. Ala177 and Val185 in the B subunit are located close to the C-terminal domain of the A subunit. Arg69 in the C subunit is located close to the A subunit.

Fig. 1.

Structure of human RNase H2. The PyMOL program was used to visualize the whole structure of human RNase H2 (PDB accession no. 3PUF). Mutated residues, Gly37, Asn212 and Arg291 in the A subunit and Ala177 and Val185 in the B subunit and Arg69 in the C subunit are shown.

Six variant enzymes, named A-G37S (Gly37 in the A subunit is replaced with Ser), A-N212I, A-R291H, B-A177T, B-V185G and C-R69W, were expressed in E. coli and purified from the cells. Figure 2 shows the SDS-PAGE pattern of the enzyme preparations under reducing conditions. WT and variants yielded three bands with molecular masses of 35.6, 34.2 and 18.6 kDa, corresponding to the B, A and C subunits, respectively. Figure 3 shows the CD spectroscopy of the enzyme preparations. WT and the six variants exhibited negative ellipticities at 210–250 nm. No appreciable changes were observed in each spectra between WT and variants. The results of SDS-PAGE and CD spectroscopy suggest that WT and all variants were purified as a heterotrimer to homogeneity, and that all variants did not suffer from any global or drastic structural changes.

Fig. 2.

SDS-PAGE of human RNase H2 variants under reducing conditions. Coomassie Brilliant Blue-stained 12.5% SDS-polyacrylamide gel showing marker proteins [Protein Molecular Weight Marker (Broad), Takarabio, Otsu, Japan] and purified enzyme preparations of the WT human RNase H2 and its variants.

Fig. 3.

CD spectra of human RNase H2 variants. The spectra at 210–340 nm were measured in 5 mM Tris-HCl buffer (pH 8.3), 20 mM KCl, 5% glycerol at 25°C with protein concentrations of 1.0 μM. Part figure shows the spectra at 210–250 nm.

Effects of mutation on the RNase H2 activity

We analysed the effects of mutation on the RNase H2 activity by the fluorescence-based activity assay with R1/D18 (Fig. 4A) and R18/D18 (Fig. 4D) as the substrate. R1/D18 emits fluorescence when it is cleaved at the 5’-end of the ribonucleotide, and the fluorescein-labelled RNA₁-DNA₃ dissociates from DNA₁₈. R18/D18 emits fluorescence when RNA₁₈ is cleaved at a site close to the 3’ end and the fluorescein-labelled RNA fragment dissociates from DNA₁₈. The activity was expressed by the initial reaction rate, which was obtained by following the increase in fluorescence intensity of the reaction solution. The dependences on the enzyme concentration of the activity are shown in Fig. 4B and C for the hydrolysis of R1/D18 and in Fig. 4E and F for the hydrolysis of R18/D18. The activities of WT and all variants increased linearly with increasing enzyme concentration. The activities of A-G37S were markedly lower than those of WT and the other five variants.

Fig. 4.

Dependence of activity of human RNase H2 variants on the enzyme concentration. (A) Sequences of R1/D18. (B) Hydrolysis of R1/D18 by A-N212I, A-R291H, B-A177T, B-V185G and C-R69W. (C) Hydrolysis of R1/D18 by A-G37S. (D) Sequences of R18/D18. (E) Hydrolysis of R18/D18 by A-N212I, A-R291H, B-A177T, B-V185G and C-R69W. (F) Hydrolysis of R18/D18 by A-G37S. The reaction was carried out in 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl₂, 60 mM KCl at 25°C.

Table I shows summary of the results shown in Fig. 4. The relative activities, defined as the ratio of the activity to that of WT, of A-G37S were markedly decreased for the hydrolysis of R1/D18 and R18/D18 (0.01 and 0.003, respectively), while those of other five variants were close to 1 (0.51–1.0 for R1/D18 and 0.71–1.2 for R18/D18). Our results were almost concordant with the results previously reported (Table II) except for A-G37S. In Coffin et al. (17), the relative activities of A-G37S were 0.3 both for the hydrolysis of DNA₁₆-RNA₄-DNA₁₀/DNA₃₀ (4-ribo) and RNA₂₀-DNA₁₀/DNA₃₀ (20-ribo). They expressed the activities by the amounts of cleaved fragment during the incubation of the enzyme with 200 nM 4-ribo or 20-ribo at 25°C for 20 min, which were assessed by gel electrophoresis of the reaction products. We expressed the activities by the increase in fluorescence intensity of the reaction solution during the incubation of the enzyme with 5.6 nM R18/D18 at 25°C. We speculate that the discrepancy might be explained by the lowered affinity of A-G37S to the substrate and the difference in substrate concentrations in the reaction.

Table I.

Activity of RNase H2 variants

	R1/D18 hydrolysisa	R18/D18 hydrolysisa	B/A
	(vo/[E]o) × 1000 (s-1)	(vo/[E]o) × 1000 (s-1)
	(A)	(B)
WT	111 (1.0)b	177 (1.0)	1.6 (1.0)
A-G37S	1.0 (0.01)	0.3 (0.003)	0.33 (0.19)
A-N212I	73 (0.66)	144 (0.81)	2.0 (1.3)
A-R291H	57 (0.51)	212 (1.2)	3.7 (2.3)
B-A177T	109 (0.98)	127 (0.72)	1.2 (0.75)
B-V185G	82 (0.74)	127 (0.72)	1.5 (0.93)
C-R69W	112 (1.0)	125 (0.71)	1.1 (0.70)

^aThe reaction was carried out in 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl₂, 60 mM KCl, 5.6 nM R1/D18 or 5.6 nM R18/D18 at 25°C.

^bNumbers in parentheses indicate values relative to WT.

Table II.

Activity and stability of RNase H2 variants previously reported

Substrate used for measuring the activity
	p(rA)/p(dT)a	1-ribob	4-riboc	20-ribod	18-riboe	ΔTm (°C)f
A-G37S	< 0.01g (5)h	< 0.01 (5), 0.005 (17)	0.3 (17)	0.3 (17)
A-E75Q/A-E294K		0.6 (29)				–1 (29)
A-R108W		0.3 (17)	0.5 (17)	1 (17)
A-A121T		1 (29)				0 (29)
A-V133M		0.4 (30)			0.1 (30)	–1.3 (30)
A-P158S		0.5 (30)			0.6 (30)	–1.8 (30)
A-A178V		< 0.01 (29)				–1 (29)
A-R186W		0.006 (17)	0.1 (17)	0.1 (17)
A-L202S		0.7 (29)				0 (29)
A-L202S/A-D205E		0.5 (29)				0 (29)
A-N212I		1 (17)	1 (17)	1 (17)
A-K221R		0.7 (29)				0 (29)
A-I244V		0.5 (29)		0 (29)
A-F230L		0.8 (17)	0.3 (17)	0.3 (17)
A-R235Q		0.003 (17)	0.002 (17)	0.001 (17)
A-T240M		0.1 (17)	1 (17)	2 (17)
A-R280G		0.9 (30)			1 (30)	–0.5 (30)
A-R291H		0.05 (6), 2 (17)	2 (17)	2 (17)		–0.7 (6)
B-G10R		0.4 (29)				–1 (29)
B-F95L		0.4 (29)				–9 (29)
B-D105A		0.3 (29)				–1 (29)
B-K162T	1 (5)	1.1 (5)
B-A177T	1 (5)	0.6 (5), 1 (6), 1 (29)				–3 (6, 29)
B-V185G	0.9 (5)	0.8 (5)
B-K233Q		0.2 (29)				–1 (29)
B-K248N		0.1 (29)				–1 (29)
B-T280A		0.9 (30)			1 (30)	–0.5 (30)
B-A287S		0.2 (29)				0 (29)
C-R69W	0.4 (5)	0.3 (5), 0.3 (6)				–2.6 (6)
C-P76L		0.8 (6)				–3 (6)
C-K90del		0.9 (29)				–1 (29)
C-E110P		0.9 (29)				–1 (29)
C-P138L		1 (6)				–2.4 (6)
C-K143I	0.9 (5)	0.7 (5), 0.1 (6)				–0.8 (6)
C-R145C		0.6 (30)			0.7 (30)	–0.5 (30)
C-P151S		0.1 (6)				–3.3 (6)

Notes: Variants characterized in this study are marked in bold.

^apoly(rA)/poly(dT).

^bDNA₁₂-RNA₁-DNA₂₇/DNA₄₀ (5), DNA₁₄-RNA₁-DNA₃/DNA₁₈ (6, 24), DNA₁₉-RNA₁-DNA₁₀/DNA₃₀ (17).

^cDNA₁₆-RNA₄-DNA₁₀/DNA₃₀.

^dRNA₂₀-DNA₁₀/DNA₃₀.

^eRNA₁₈/DNA₁₈.

^fΔT_m [= (T_m of variants)–(T_m of WT)] in the fluorescence-based thermal stability assay.

^gThe activity compared to that of the WT.

^hNumbers in parentheses indicate references.

The dependence of the reaction rate on the substrate concentration was examined with respect to WT and R18/D18 (Supplementary Fig. S1). As described in the legend to Supplementary Fig. S1, to avoid the inner-filter effect of the substrate, the reaction was stopped at an appropriate time by adding EDTA. Then, the reaction solution was diluted, and the fluorescence intensity was measured. The reaction rate increased linearly against the R18/D18 concentration (0–10 μM) and did not exhibit a saturated profile of the Michaelis–Menten kinetics. This result indicated that Michaelis–Menten kinetics was not applicable, and k_cat and K_m values were not separately determined.

Effects of AGS-causing mutation on the salt-dependence of RNase H2 activity

We previously analysed the effects of neutral salts on the activity of WT in the hydrolysis of R1/D18 and R18/D18 (21): NaCl, KCl, RbCl and NaBr increased the activity to 170–390%, while LiCl, LiBr and CsCl inhibited it, suggesting that species of cation, but not anion, is responsible for activity. We analysed the effects of NaCl on the hydrolysis of R1/D18 (Fig. 5A) and R18/D18 (Fig. 5B), the effects of KCl on the hydrolysis of R1/D18 (Fig. 5C) and R18/D18 (Fig. 5D) and the effects of CsCl on the hydrolysis of R1/D18 (Fig. 5E) and R18/D18 (Fig. 5F). Relative activity of RNase H2 was defined as the ratio of the activity in the presence of salt to that in the absence. The relative activities of WT and all variants were the highest at 20 mM NaCl (350% for WT and 100–250% for variants in the hydrolysis of R1/D18 (Fig. 5A), 500% for WT and 110–580% for variants in the hydrolysis of R18/D18 (Fig. 5B), 170% for WT and 110–270% for variants in the hydrolysis of R1/D18 (Fig. 5C) and 400% for WT and 190–320% for variants in the hydrolysis of R18/D18 (Fig. 5D) and decreased with increasing NaCl or KCl concentrations. These results indicated that all variants exhibited relatively similar NaCl-and KCl-dependences of activity to WT, but the profiles were different between the hydrolysis of R1/D18 and R18/D18. Considering that potassium ion is abundant intracellularly, our results suggested that it might inhibit the RNase H2 activity in cells to some extent. The relative activities of WT and all variants except for A-G37S were stable or slightly increased at 0–60 mM CsCl in the hydrolysis of R1/D18 (Fig. 5E) and at 0–20 mM CsCl in the hydrolysis of R18/D18 (Fig. 5F) and decreased with increasing CsCl concentrations. A-G37S exhibited more rapid decrease than WT and other variants. These results indicated that all variants except for A-G37S exhibited similar CsCl-dependences of activity to WT, but the profiles were different between the hydrolysis of R1/D18 and R18/D18 like the case with the NaCl-dependences.

Fig. 5.

Dependence of activity of human RNase H2 variants on salt concentration. (A) Hydrolysis of R1/D18 at 0–200 mM NaCl. (B) Hydrolysis of R18/D18 at 0–200 mM NaCl. (C) Hydrolysis of R1/D18 at 0–200 mM KCl. (D) Hydrolysis of R18/D18 at 0–200 mM KCl. (E) Hydrolysis of R1/D18 at 0–200 mM CsCl. (F) Hydrolysis of R18/D18 at 0–200 mM CsCl. The reaction was carried out in 50 mM Tris-HCl buffer (pH 8.0), 5 mM MgCl₂, 0–200 mM NaCl, KCl, or CsCl at 25°C.

From these results, we speculate that the salt-based activation and inhibition of human RNase H2 might result from not only the interaction between respective ions and particular residues but also the interaction between respective ions and the RNA/DNA hybrid. In DNA synthesis by DNA polymerase, two Mg²⁺ ions bind the enzyme whereas another Mg²⁺ ion binds the α-phosphate oxygen of the incoming dNTP (24, 25). This third Mg²⁺ is thought to be involved in fidelity (26). We reported the effects of Mg²⁺concentrations on the RNA-dependent DNA synthesis by reverse transcriptases were different from those on the DNA-dependent DNA synthesis (27). These evidences support our speculation that the neutral salts might affect RNase H2 activity partly by interacting with the RNA/DNA hybrid.

Effects of AGS-causing mutation on RNase H2 stability

To analyse the effects of AGS-causing mutation on the RNase H2 stability, we performed CD-based assay by monitoring θ₂₂₂ in the range of 30–70°C (Fig. 6). The denaturation curves of WT and variants showed apparent two-state model as expressed in Scheme 1

$$N\rightleftarrows D$$ (Scheme \ 1)

where N and D represent the native and denatured species, respectively. Fraction unfolded (F_u) was determined after normalizing θ₂₂₂ of native and denatured RNase H2 between 0 and 1, according to Eq. 1.

$$F_{\mathrm{u}}=\left(A_{\mathrm{O}}-A_{\mathrm{N}}\right)/\left(A_{\mathrm{D}}-A_{\mathrm{N}}\right)$$ (Eq. \ 1)

where A_O is the observed θ₂₂₂ of RNase H2 at various temperatures, and A_N and A_D are θ₂₂₂ of native and denatured enzymes, respectively. The melting temperature (T_m) was defined as the one at which F_u is 0.5. The T_m values were 56°C for WT, 53°C for A-N212I, A-R291H, B-A177T, B-V185G and C-R69W and 50°C for A-G37S. These results indicated that all six AGS-causing mutations decreased the stability. The marked decrease in A-G37S was contrary to that glycine replacement is one of general strategies in protein engineering to increase protein stability. Our results suggest that Gly37 in the A subunit plays an important role not only in activity but also in stability.

Fig. 6.

Thermal denaturation of human RNase H2 variants. θ₂₂₂ of WT and variants were monitored from 30 to 70°C at 1°C/min. (A) WT, A-G37S, A-N212I and A-R291H. Their melting temperatures are 56, 50, 53 and 53°C, respectively. (B) WT, B-A177T, B-V185G and C-R69W. Their melting temperatures are 56, 53, 53 and 53°C, respectively.

According to Günther et al. (27), the fluorescence-based thermal shift assay (ThermoFluor Assay) revealed that the T_m values of human RNase H2 variants B-F95L and B-A177T were lower by 9°C and 3°C, respectively, than that of WT, while those of other 13 variants were similar to that of WT (Table II). In this study, the T_m values of all six variants were lower by 3–6°C than that of WT. This discrepancy might be due to the difference in the mechanism to detect denaturation between the two methods, the binding of dye to hydrophobic patches in the thermal shift assay and the detection of α helix in CD.

Effects of AGS-causing mutation on the dissociation of RNase H2 subunits

We previously reported that in gel filtration chromatography (GFC) of the purified enzyme preparation, WT was eluted as a single peak at the retention time corresponding to a molecular mass of the heterotrimer (88 kDa) (22). We examined if AGS-causing mutations affected the heterotrimer-forming stability under the condition that the enzyme concentration is low. Figure 7 shows the GFC elution pattern of WT and variants together with the SDS-PAGE patterns of the GFC fractions. In GFC, l-Arg (0.5 M) was contained in the equilibrium buffer to improve resolution (28), although WT and all variants of RNase H2 did not exhibit the activity in the presence of 0.5 M l-Arg. When 8.6 μg of WT was applied, a single peak appeared at the retention time of 15.3–16.5 min. The peak fractions contained all A, B and C subunits, suggesting that WT was eluted as a heterotrimer. Same results were obtained with other five variants except for A-R291H. When 8.6 μg of A-R291H was applied, two peaks appeared at the retention time of 15.3–17.7 and 17.7–18.7 min, respectively. The first peak fractions contained all three subunits, while the second peak contained only the A subunit. The RNase H2 assay of the two peak fractions by the fluorescence-based activity assay with R1/D18 or R18/D18 showed that the first peak had the activity while the second peak did not (Supplementary Fig. S2). These results suggested that in A-R291H, the heterotrimer dissociated into the A subunit and, presumably, the complex of the B and C subunits. The presence of the complex of the B and C subunits was previously suggested in the co-expression of the A subunit which lacked the C-terminal and the intact B and C subunits (6). When 2.9 μg of enzyme was applied, multiple peaks appeared in A-G37S, A-R291H, B-A177T and B-V185G. On the other hand, when 26 μg of enzyme was applied, such multiple peaks were less obvious, indicating that dissociation depended on enzyme concentration. These results suggested that the AGS-causing mutations decrease the heterotrimer-forming stability.

Fig. 7.

Elution patterns of gel filtration column chromatography of human RNase H2 variants. WT and variants (26, 8.6, or 2.9 μg) were applied onto the column. Silver-stained 12.5% SDS-polyacrylamide gel is also shown.

Conclusion

AGS-causing mutations affected the activity and stability of human RNase H2 with varying degrees depending on mutation species. A-G37S exhibited markedly lower activity and stability than other variants, while other five variants had decreased stability rather than decreased activity. Uehara et al. showed that even G37S was active enough for mouse embryo to survive to birth (10). We recently showed that RNase H2 gene knockout NIH3T3 cells (KO cells) lacked a single ribonucleotide excision activity and contained more ribonucleotides in genomic DNA than the WT NIH3T3 cells (13). However, unlike the mouse embryonic fibroblasts isolated from inducible RNase H2 knockout mice (14), the KO cells did not exhibit several tens of times higher expression of interferon-stimulated genes (13). Construction of mammalian cells possessing the AGS-causing mutation in the RNase H2 gene and characterization of the cellular events of the cells and the activity and stability of the RNase H2 variants expressed in the cells are required for exploring the effects of the mutation on cellular events.

Funding

This study was supported in part by Grants-in-Aid for Scientific Research (no. 21580110 for K.K., K.Y. and no. 18J14339 for M.B.) from the Japan Society for the Promotion of Science (K.K., K.Y.), the Salt Science Research Foundation (K.K., K.Y.), and the IRP of NICHD (R.J.C.).

Conflict of Interest

None declared.

Glossary

Abbreviations

AGS

Aicardi-Goutières syndrome

Dabcyl

4-((4-(dimethylamino)-phenyl)-azo)-benzoic acid

DTT

dithiothreitol

FITC

fluorescein-5-isothiocynate

IPTG

isopropyl β-d-1 thiogalactopyranoside