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.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.     

PCNA directs type 2 RNase H activity on DNA replication and repair substrates

Ribonuclease H2 is the major nuclear enzyme degrading cellular RNA/DNA hybrids in eukaryotes and the sole nuclease known to be able to hydrolyze ribonucleotides misincorporated during genomic replication. Mutation in RNASEH2 causes Aicardi-Goutières syndrome, an auto-inflammatory disorder that may arise from nucleic acid byproducts generated during DNA replication. Here, we report the crystal structures of Archaeoglobus fulgidus RNase HII in complex with PCNA, and human PCNA bound to a C-terminal peptide of RNASEH2B. In the archaeal structure, three binding modes are observed as the enzyme rotates about a flexible hinge while anchored to PCNA by its PIP-box motif. PCNA binding promotes RNase HII activity in a hinge-dependent manner. It enhances both cleavage of ribonucleotides misincorporated in DNA duplexes, and the comprehensive hydrolysis of RNA primers formed during Okazaki fragment maturation. In addition, PCNA imposes strand specificity on enzyme function, and by localizing RNase H2 and not RNase H1 to nuclear replication foci in vivo it ensures that RNase H2 is the dominant RNase H activity during nuclear replication. Our findings provide insights into how type 2 RNase H activity is directed during genome replication and repair, and suggest a mechanism by which RNase H2 may suppress generation of immunostimulatory nucleic acids.     

Genome instability consequences of RNase H2 Aicardi-Goutières syndrome alleles

The RNase H2 complex is a conserved heterotrimeric enzyme that degrades RNA:DNA hybrids and promotes excision of rNMPs misincorporated during DNA replication. Failure to remove ribonucleotides from DNA leads to genomic instability in yeast and humans. The monogenic Aicardi-Goutières syndrome (AGS) results from mutation in one of several genes, among which are those encoding the RNase H2 subunits. The complete cellular and genomic consequences of RNASEH2 mutations and the precise connection to disease remain unclear. To learn more about the effect of RNASEH2 mutations on the cell, we used yeast as a model of AGS disease. We have generated yeast strains bearing AGS-associated mutations in RNASEH2 genes. There is a range of disease presentation in patients bearing these RNASEH2 variants. Here we report on in vivo phenotypes of genomic instability, including mutation and recombination rates, and synthetic gene interactions. These phenotypes provide insight into molecular consequences of RNASEH2 mutations, and lay the groundwork for further study of genomic instability as a contributing factor to AGS disease.     

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.     

Contributions of the two accessory subunits,RNASEH2B and RNASEH2C,to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.     

Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H.

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene.     

Mitochondrial RNase H1 activity regulates R-loop homeostasis to maintain genome integrity and enable early embryogenesis in Arabidopsis

Plant mitochondrial genomes undergo frequent homologous recombination (HR). Ectopic HR activity is inhibited by the HR surveillance pathway, but the underlying regulatory mechanism is unclear. Here, we show that the mitochondrial RNase H1 AtRNH1B impairs the formation of RNA:DNA hybrids (R-loops) and participates in the HR surveillance pathway in Arabidopsis thaliana. AtRNH1B suppresses ectopic HR at intermediate-sized repeats (IRs) and thus maintains mitochondrial DNA (mtDNA) replication. The RNase H1 AtRNH1C is restricted to the chloroplast; however, when cells lack AtRNH1B, transport of chloroplast AtRNH1C into the mitochondria secures HR surveillance, thus ensuring the integrity of the mitochondrial genome and allowing embryogenesis to proceed. HR surveillance is further regulated by the single-stranded DNA-binding protein ORGANELLAR SINGLE-STRANDED DNA BINDING PROTEIN1 (OSB1), which decreases the formation of R-loops. This study uncovers a facultative dual targeting mechanism between organelles and sheds light on the roles of RNase H1 in organellar genome maintenance and embryogenesis.

This study clarifies the function of mitochondrial RNase H1 in genome stability and early embryogenesis in plants, and shows that mitochondrial R-loops are involved in homologous recombination surveillance of mtDNA. Facultative re-targeting of the chloroplast RNase H1 protein to mitochondria, in response to cellular conditions, can help guarantee mitochondrial RNase H1 activity.     

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.     

RnhP is a plasmid‐borne RNase HI that contributes to genome maintenance in the ancestral strain Bacillus subtilis NCIB 3610

RNA‐DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA‐DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA‐DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper‐replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid‐encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper‐replication.     

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.     

Junction ribonuclease: a ribonuclease HII orthologue from Thermus thermophilus HB8 prefers the RNA-DNA junction to the RNA/DNA heteroduplex

The genome of an extremely thermophilic bacterium, Thermus thermophilus HB8, contains a single ORF (open reading frame) encoding an RNase-HII-like sequence. Despite the presence of significant amino acid sequence identities with RNase (ribonuclease) HII enzymes, the ORF TTHA0198 could not suppress the temperature-sensitive growth defect of an RNase-H-deficient Escherichia coli mutant and the purified recombinant protein could not cleave an RNA strand of an RNA/DNA heteroduplex, suggesting that the TTHA0198 exhibited no RNase H activity both in vivo and in vitro. When oligomeric RNA-DNA/DNAs were used as a mimic substrate for Okazaki fragments, however, the protein cleaved them only at the 5' side of the last ribonucleotide at the RNA-DNA junction. In fact, the TTHA0198 protein prefers the RNA-DNA junction to the RNA/DNA hybrid. We have referred to this activity as JRNase (junction RNase) activity, which recognizes an RNA-DNA junction of the RNA-DNA/DNA heteroduplex and cleaves it leaving a mono-ribonucleotide at the 5' terminus of the RNA-DNA junction. E. coli and Deinococcus radiodurans RNases HII also cleaved the RNA-DNA/DNA substrates at the same site with a different metal-ion preference from that for RNase H activity, implying that the enzymes have JRNase activity as well as RNase H activity. The specialization in the JRNase activity of the RNase HII orthologue from T. thermophilus HB8 (Tth-JRNase) suggests that the JRNase activity of RNase HII enzymes might be independent of the RNase H activity.