The structural and biochemical characterization of human RNase H2 complex reveals the molecular basis for substrate recognition and Aicardi-Goutières syndrome defects

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5′)RNA-DNA(3′)/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5′)RNA-DNA(3′) junction and very poorly cleave RNA/DNA hybrids in the presence of Mg²⁺ ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.     

Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage

Two classes of RNase H hydrolyze RNA of RNA/DNA hybrids. In contrast to RNase H1 that requires four ribonucleotides for cleavage, RNase H2 can nick duplex DNAs containing a single ribonucleotide, suggesting different in vivo substrates. We report here the crystal structures of a type 2 RNase H in complex with substrates containing a (5')RNA-DNA(3') junction. They revealed a unique mechanism of recognition and substrate-assisted cleavage. A conserved tyrosine residue distorts the nucleic acid at the junction, allowing the substrate to function in catalysis by participating in coordination of the active site metal ion. The biochemical and structural properties of RNase H2 explain the preference of the enzyme for junction substrates and establish the structural and mechanistic differences with RNase H1. Junction recognition is important for the removal of RNA embedded in DNA and may play an important role in DNA replication and repair.     

Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain

Ribonucleases H have mostly been implicated in eliminating short RNA primers used for initiation of lagging strand DNA synthesis. Escherichia coli RNase HI cleaves these RNA-DNA hybrids in a distributive manner. We report here that eukaryotic RNases H1 have evolved to be processive enzymes by attaching a duplex RNA-binding domain to the RNase H region. Highly conserved amino acids of the duplex RNA-binding domain are required for processivity and nucleic acid binding, which leads to dimerization of the protein. The need for a processive enzyme underscores the importance in eukaryotic cells of processing long hybrids, most of which remain to be identified. However, long RNA-DNA hybrids formed during immunoglobulin class-switch recombination are potential targets for RNase H1 in the nucleus. In mitochondria, where RNase H1 is essential for DNA formation during embryogenesis, long hybrids may be involved in DNA replication.     

Structural biochemistry of a type 2 RNase H: RNA primer recognition and removal during DNA replication

DNA replication and cellular survival requires efficient removal of RNA primers during lagging strand DNA synthesis. In eukaryotes, RNA primer removal is initiated by type 2 RNase H, which specifically cleaves the RNA portion of an RNA-DNA/DNA hybrid duplex. This conserved type 2 RNase H family of replicative enzymes shares little sequence similarity with the well-characterized prokaryotic type 1 RNase H enzymes, yet both possess similar enzymatic properties. Crystal structures and structure-based mutational analysis of RNase HII from Archaeoglobus fulgidus, both with and without a bound metal ion, identify the active site for type 2 RNase H enzymes that provides the general nuclease activity necessary for catalysis. The two-domain architecture of type 2 RNase H creates a positively charged binding groove and links the unique C-terminal helix-loop-helix cap domain to the active site catalytic domain. This architectural arrangement apparently couples directional A-form duplex binding, by a hydrogen-bonding Arg-Lys phosphate ruler motif, to substrate-discrimination, by a tyrosine finger motif, thereby providing substrate-specific catalytic activity. Combined kinetic and mutational analyses of structurally implicated substrate binding residues validate this binding mode. These structural and mutational results together suggest a molecular mechanism for type 2 RNase H enzymes for the specific recognition and cleavage of RNA in the RNA-DNA junction within hybrid duplexes, which reconciles the broad substrate binding affinity with the catalytic specificity observed in biochemical assays. In combination with a recent independent structural analysis, these results furthermore identify testable molecular hypotheses for the activity and function of the type 2 RNase H family of enzymes, including structural complementarity, substrate-mediated conformational changes and coordination with subsequent FEN-1 activity.     

RNA-DNA hybrids containing damaged DNA are substrates for RNase H

During the replication of the lagging strand, RNA-DNA hybrids are formed and the RNA is subsequently degraded by the action of RNase H. Little is known about the effects of damaged DNA on lagging strand replication and subsequent RNA removal. The rates and sites of digestion by E. coli RNase H of RNA-DNA hybrids containing either a thymine glycol or urea site in the DNA strand have been examined. The cleavage patterns for duplexes containing thymine glycol or urea differ from that of a fully complementary duplex. There is one major product of the digestion of the fully complementary hybrid, but three products are formed in the reactions with the hybrids containing damaged DNAs. Cleavage is partially redirected to the position adjacent to the damaged sites. The overall rate of cleavage of these hybrids containing damaged DNA is comparable to that of the fully complementary duplex. These results indicate that the cleavage of RNA-DNA hybrids by RNase H is less selective when a damaged site is present in the DNA strand.     

RNase H is an exo- and endoribonuclease with asymmetric directionality,depending on the binding mode to the structural variants of RNA:DNA hybrids

RNase H is involved in fundamental cellular processes and is responsible for removing the short stretch of RNA from Okazaki fragments and the long stretch of RNA from R-loops. Defects in RNase H lead to embryo lethality in mice and Aicardi-Goutieres syndrome in humans, suggesting the importance of RNase H. To date, RNase H is known to be a non-sequence-specific endonuclease, but it is not known whether it performs other functions on the structural variants of RNA:DNA hybrids. Here, we used Escherichia coli RNase H as a model, and examined its catalytic mechanism and its substrate recognition modes, using single-molecule FRET. We discovered that RNase H acts as a processive exoribonuclease on the 3′ DNA overhang side but as a distributive non-sequence-specific endonuclease on the 5′ DNA overhang side of RNA:DNA hybrids or on blunt-ended hybrids. The high affinity of previously unidentified double-stranded (ds) and single-stranded (ss) DNA junctions flanking RNA:DNA hybrids may help RNase H find the hybrid substrates in long genomic DNA. Our study provides new insights into the multifunctionality of RNase H, elucidating unprecedented roles of junctions and ssDNA overhang on RNA:DNA hybrids.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Complicated water orientations in the minor groove of the B-DNA decamer d(CCATTAATGG)2 observed by neutron diffraction measurements

It has long been suspected that the structure and function of a DNA duplex can be strongly dependent on its degree of hydration. By neutron diffraction experiments, we have succeeded in determining most of the hydrogen (H) and deuterium (D) atomic positions in the decameric d(CCATTAATGG)₂ duplex. Moreover, the D positions in 27 D₂O molecules have been determined. In particular, the complex water network in the minor groove has been observed in detail. By a combined structural analysis using 2.0 Å resolution X-ray and 3.0 Å resolution neutron data, it is clear that the spine of hydration is built up, not only by a simple hexagonal hydration pattern (as reported in earlier X-ray studies), but also by many other water bridges hydrogen-bonded to the DNA strands. The complexity of the hydration pattern in the minor groove is derived from an extraordinary variety of orientations displayed by the water molecules.     

Insights into RNA/DNA hybrid recognition and processing by RNase H from the crystal structure of a non-specific enzyme-dsDNA complex

Ribonuclease HI (RNase H) is a member of the nucleotidyl-transferase superfamily and endo-nucleolytically cleaves the RNA portion in RNA/DNA hybrids and removes RNA primers from Okazaki fragments. The enzyme also binds RNA and DNA duplexes but is unable to cleave either. Three-dimensional structures of bacterial and human RNase H catalytic domains bound to RNA/DNA hybrids have revealed the basis for substrate recognition and the mechanism of cleavage. In order to visualize the enzyme’s interactions with duplex DNA and to establish the structural differences that afford tighter binding to RNA/DNA hybrids relative to dsDNA, we have determined the crystal structure of Bacillus halodurans RNase H in complex with the B-form DNA duplex [d(CGCGAATTCGCG)]2. The structure demonstrates that the inability of the enzyme to cleave DNA is due to the deviating curvature of the DNA strand relative to the substrate RNA strand and the absence of Mg2+ at the active site. A subset of amino acids engaged in contacts to RNA 2′-hydroxyl groups in the substrate complex instead bind to bridging or non-bridging phosphodiester oxygens in the complex with dsDNA. Qualitative comparison of the enzyme’s interactions with the substrate and inhibitor duplexes is consistent with the reduced binding affinity for the latter and sheds light on determinants of RNase H binding and cleavage specificity.     

Junction ribonuclease activity specified in RNases HII/2

Junction ribonuclease (JRNase) recognizes the transition from RNA to DNA of an RNA-DNA/DNA hybrid, such as an Okazaki fragment, and cleaves it, leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. Although this JRNase activity was originally reported in calf RNase H2, some other RNases H have recently been suggested to possess it. This paper shows that these enzymes can also cleave an RNA-DNA/RNA heteroduplex in a manner similar to the RNA-DNA/DNA substrate. The cleavage site of the RNA-DNA/RNA substrate corresponds to the RNA/RNA duplex region, indicating that the cleavage activity cannot be categorized as RNase H activity, which specifically cleaves an RNA strand of an RNA/DNA hybrid. Examination of several RNases H with respect to JRNase activity suggested that the activity is only found in RNase HII orthologs. Therefore, RNases HIII, which are RNase HII paralogs, are distinguished from RNases HII by the absence of JRNase activity. Whether a substrate can be targeted by JRNase activity would depend only on whether or not an RNA-DNA junction consisting of one ribonucleotide and one deoxyribonucleotide is included in the duplex. In addition, although the activity has been reported not to occur on completely single-stranded RNA-DNA, it can recognize a single-stranded RNA-DNA junction if a double-stranded region is located adjacent to the junction.     

Crystal structure of RNase H3–substrate complex reveals parallel evolution of RNA/DNA hybrid recognition

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA–DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2′-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA.     

Structural basis of cleavage by RNase H of hybrids of arabinonucleic acids and RNA

The origins of the substrate specificity of Escherichia coli RNase H1 (termed RNase H here), an enzyme that hydrolyzes the RNA strand of DNA-RNA hybrids, are not understood at present. Although the enzyme binds double-stranded RNA, no cleavage occurs with such duplexes [Lima, W. F., and Crooke, S. T. (1997) Biochemistry 36, 390]. Therefore, the hybrid substrates may not adopt a canonical A-form geometry. Furthermore, RNase H is exquisitely sensitive to chemical modification of the DNA strands in hybrid duplexes. This is particularly relevant to the RNase H-dependent pathway of antisense action. Thus, only very few of the modifications currently being evaluated as antisense therapeutics are tolerated by the enzyme, among them phosphorothioate DNA (PS-DNA). Recently, hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'F-ANA analogue were shown to be substrates of RNase H [Damha, M. J., et al. (1998) J. Am. Chem. Soc. 120, 12976]. Using X-ray crystallography, we demonstrate here that ANA analogues, such as 2'F-ANA [Berger, I., et al. (1998) Nucleic Acids Res. 26, 2473] and [3.3.0]bicyclo-ANA (bc-ANA), may not be able to adopt sugar puckers that are compatible with pure A- or a B-form duplex geometries, but rather prefer the intermediate O4'-endo conformation. On the basis of the observed conformations of these ANA analogues in a DNA dodecamer duplex, we have modeled a duplex of an all-C3'-endo RNA strand and an all-O4'-endo 2'F-ANA strand. This duplex exhibits a minor groove width that is intermediate between that of A-form RNA and B-form DNA, a feature that may be exploited by the enzyme in differentiating between RNA duplexes and DNA-RNA hybrids. Therefore, the combination of the established structural and functional properties of ANA analogues helps settle existing controversies concerning the discrimination of substrates by RNase H. Knowlegde of the structure of an analogue that exhibits enhanced RNA affinity while not interfering with RNase H activity may prove helpful in the design of future antisense modifications.     

Properties of cloned and expressed human RNase H1.

We have characterized cloned His-tag human RNase H1. The activity of the enzyme exhibited a bell-shaped response to divalent cations and pH. The optimum conditions for catalysis consisted of 1 mM Mg(2+) and pH 7-8. In the presence of Mg(2+), Mn(2+) was inhibitory. Human RNase H1 shares many enzymatic properties with Escherichia coli RNase H1. The human enzyme cleaves RNA in a DNA-RNA duplex resulting in products with 5'-phosphate and 3'-hydroxy termini, can cleave overhanging single strand RNA adjacent to a DNA-RNA duplex, and is unable to cleave substrates in which either the RNA or DNA strand has 2' modifications at the cleavage site. Human RNase H1 binds selectively to "A-form"-type duplexes with approximately 10-20-fold greater affinity than that observed for E. coli RNase H1. The human enzyme displays a greater initial rate of cleavage of a heteroduplex-containing RNA-phosphorothioate DNA than an RNA-DNA duplex. Unlike the E. coli enzyme, human RNase H1 displays a strong positional preference for cleavage, i.e. it cleaves between 8 and 12 nucleotides from the 5'-RNA-3'-DNA terminus of the duplex. Within the preferred cleavage site, the enzyme displays modest sequence preference with GU being a preferred dinucleotide. The enzyme is inhibited by single-strand phosphorothioate oligonucleotides and displays no evidence of processivity. The minimum RNA-DNA duplex length that supports cleavage is 6 base pairs, and the minimum RNA-DNA "gap size" that supports cleavage is 5 base pairs.     

Pulse Dipolar ESR of Doubly Labeled Mini TAR DNA and Its Annealing to Mini TAR RNA

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3′- and 5′-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26–27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ∼35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.     

Targeting degradation of RNA by RNase H using DNA hairpins

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.     

The solution structure of [d(CGC)r(aaa)d(TTTGCG)](2): hybrid junctions flanked by DNA duplexes

The solution structure and hydration of the chimeric duplex [d(CGC)r(aaa)d(TTTGCG)]₂, in which the central hybrid segment is flanked by DNA duplexes at both ends, was determined using two-dimensional NMR, simulated annealing and restrained molecular dynamics. The solution structure of this chimeric duplex differs from the previously determined X-ray structure of the analogous B-DNA duplex [d(CGCAAATTTGCG)]₂ as well as NMR structure of the analogous A-RNA duplex [r(cgcaaauuugcg)]₂. Long-lived water molecules with correlation time τ_c longer than 0.3 ns were found close to the RNA adenine H2 and H1′ protons in the hybrid segment. A possible long-lived water molecule was also detected close to the methyl group of 7T in the RNA–DNA junction but not with the other two thymines (8T and 9T). This result correlates with the structural studies that only DNA residue 7T in the RNA–DNA junction adopts an O4′-endo sugar conformation, while the other DNA residues including 3C in the DNA–RNA junction, adopt C1′-exo or C2′-endo conformations. The exchange rates for RNA C2′-OH were found to be ~5–20 s^–1. This slow exchange rate may be due to the narrow minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂, which may trap the water molecules and restrict the dynamic motion of hydroxyl protons. The minor groove width of [d(CGC)r(aaa)d(TTTGCG)]₂ is wider than its B-DNA analog but narrower than that of the A-RNA analog. It was further confirmed by its titration with the minor groove binding drug distamycin. A possible 2:1 binding mode was found by the titration experiments, suggesting that this chimeric duplex contains a wider minor groove than its B-DNA analog but still narrow enough to hold two distamycin molecules. These distinct structural features and hydration patterns of this chimeric duplex provide a molecular basis for further understanding the structure and recognition of DNA·RNA hybrid and chimeric duplexes.