Physiological, Biochemical, and Molecular-Genetic Events Governing the Recognition and Resolution of RNA/DNA Hybrids
- Robert J. Crouch, PhD, Head, Section on Formation of RNA
- Susana M. Cerritelli, PhD, Staff Scientist
- Naushaba Hasin, PhD, Postdoctoral Fellow
- Ryo Uehara, PhD, Postdoctoral Fellow
- Kiran Sakhuja, MS, MSc, Biologist
Damaged DNA is one of the leading causes of many human diseases and disorders. We study the formation and resolution of RNA/DNA hybrids, which occur during DNA and RNA synthesis. Such hybrid molecules may lead to increased DNA damage but may also play critical roles in normal cellular processes. We are interested in how RNA/DNA hybrids are resolved and in the role that ribonucleases H (RNases H) play in their elimination (Reference 1). Two classes of RNases H, Class I and Class II, are present in most organisms. Our studies have shown that mice deleted for the Rnaseh1 gene arrest embryonic development at day 8.5 as a result of failure to amplify mitochondrial DNA. Others have found that the Aicardi-Goutières Syndrome (AGS), a severe neurological disorder with symptoms appearing at or soon after birth, can be caused by defective human RNase H2. We are examining mouse models of AGS to gain insight into the human disorder. We employ molecular-genetic and biochemical tools in yeast and mouse models in our research to understand the mechanisms and functions of RNases H and their associated substrates.
There are several types of RNA/DNA hybrids structures formed and processed differently. Simple RNA/DNA hybrids consist of one strand of RNA paired with one strand of DNA. The HIV-AIDS reverse transcriptase (RT) forms such hybrids when copying its genomic RNA into DNA. The RT also has an RNase H domain that is structurally and functionally similar to the class I cellular RNase H and is necessary for several steps of viral DNA synthesis. R-loop hybrids have two separated DNA strands, with one hybridized to RNA while the other is in single-stranded form. These structures sometimes form during transcription and can lead to chromosomal breakage. However, they are also part of the normal recombination process of switching (recombination) from one form of immunoglobulin to another, resulting in different isoforms of antibodies. Another form of hybrid are single or multiple ribonucleotides incorporated into DNA during replication (Reference 2). The first two types of hybrid are substrates for class I and II RNases H. The third is uniquely recognized by type 2 RNases H.
Contrasts between Class I and Class II RNases H
Many of our investigations over the past few years have focused on RNase H1. RNase H1 recognizes the 2′-OH of four consecutive ribonucleotides while the DNA strand is distorted to fit into a pocket of the enzyme. Thus, the enzyme requires more than one ribonucleotide for cleavage of RNA in RNA/DNA hybrids. RNases H1 consist of a single polypeptide in both eukaryotes and prokaryotes. In contrast, RNase H2 is a complex of three distinct polypeptides in eukaryotes but is a single polypeptide in prokaryotes. The catalytic subunit of the heterotrimeric RNase H2 of eukaryotes is similar in its primary amino acid sequence to the prokaryotic enzyme. RNase H2 can recognize and cleave a single ribonucleotide or the transition from the ribonucleotide in the case of RNA–primed DNA synthesis (e.g., rrrrrDDDD in DNA—italics indicate transition from ribonucleotide to deoxyribonucleotide).
Dual activities of RNase H2 and Aicardi-Goutières syndrome
Eukaryotic RNases H2 recognize and resolve RNA hybridized or covalently attached to DNA—two chemically distinct structures—using the same catalytic mechanism of hydrolysis. RNase H2 mutations that reduce catalytic activity, or fail to properly interact with in vivo substrates, cause Aicardi-Goutières syndrome (AGS). Mutations in seven genes are known to cause AGS, with more than 50% of AGS patients having mutations in any of the three subunits of RNase H2. We previously expressed (in Escherichia coli) and purified human RNases H2 with mutations corresponding to several of those seen in AGS patients. We found only one with significant loss of RNase H2 activity. Using the structure we determined, we can locate all known mutations in RNase H2 that cause AGS, and the mutation with low RNase H2 activity is located near the catalytic center of the enzyme. The wide distribution of the mutations suggests that modest changes in stability, interaction with other unknown proteins, and loss of catalysis can all cause AGS.
We are developing mouse models of AGS to clarify which defects are associated with each RNase H2 activity. Mice bearing the RNase H2A G37S mutation in homozygous form are perinatal lethal, i.e., either dead at birth or die within a few hours of birth. Mutations in another gene, TREX1, also cause AGS, and it has been shown that homozygous knockout (KO) mice are viable but die after a few weeks owing to a cardiomyopathy that can be prevented by either blocking an innate or adaptive immune response. In contrast, the G37S-mutant perinatal lethality and the fact that RNase H2 KO mice die early in embryogenesis suggest a more severe defect than that seen in TREX1-KO mice. We attempted to rescue the perinatal phenotype by eliminating one part of the innate immune pathway or by completely inactivating the adaptive immune response. These mice are no different than the innate or adaptive competent mice, suggesting the possibility of a different issue between the TREX1– and RNase H2–mutant mice. However, the expression of several interferon-stimulated genes (ISGs) is elevated in mouse embryonic fibroblasts (MEFs) derived from G37S homozygous embryos, supporting a role for innate immunity the AGS phenotype. Damaged DNA that finds its way to the cytoplasm can be sensed by the cGAS protein producing the small molecule cGAMP, which interacts with the Sting protein, an important protein for the DNA–sensing innate immune pathway. Mice that are homozygous for G37S and deleted for the Sting gene are mostly perinatal lethal but no longer exhibit increases in ISGs. Interestingly, a small fraction of the double G37S–Sting KO are viable, indicating only limited involvement of ISGs in perinatal lethality. Further studies are under way, which we expect will lead us to the cause of lethality.
To distinguish the defects that persistent RNA/DNA hybrids and single ribonucleotides joined to DNA caused in vivo, we modified RNase H2 to make an enzyme that could only cleave one type of substrate. Based on a rational design comparing the structures of RNases H2 and H3, we unlinked the two activities to yield an enzyme that processes RNA/DNA, but leaves single ribonucleoside monophosphates (rNMPs) attached to DNA. RNases H2 and H3 have similar 3D structures, but RNase H3 does not cleave single rNMPs in DNA. We first examined in vitro activities of our new RNase H2 mutant using, among others, the ribonucleotide excision repair (RER) assay recently developed by our collaborators Peter Burgers and Justin Sparks (Reference 2). The in vitro results show complete lack of removal of single ribonucleotides but only modest reduction in hydrolysis of RNA/DNA hybrids. In vivo, our RNase H2 mutant gave the signature 2–5 bp deletions in the yeast CAN1 gene associated with incorporation of rNMP into DNA in the absence of RNase H2. The mutant enzyme, which we call RED (Ribonucleotide Excision Deficient) resolves RNA/DNA hybrids, which are substrates of both RNase H1 and RNase H2. However, our RNase H2RED identified a unique set of hybrids that formed when homologous recombination is defective, owing to loss of SGS1 helicase, and that can only be processed by RNase H2, most likely because it has special access via contacts with other cell components. Thus, the synthetic defect observed in sgs1D, rnh201D strains results from problems associated with persistent R-loops. Given that our RNase H2 mutant enzyme resolves these R-loops, the synthetic defect is absent when the RNase H2RED is present in a strain deleted for SGS1 (Reference 3).
The RNaseh2a G37S mutation, which is found in a few AGS patients, results in limited activity in vitro. We investigated how the corresponding mutation in yeast RNase H2 (G42S) protein functions. In vitro it had very poor activity on RNA/DNA hybrids and on cleavage of single rNMPs in DNA. In vivo it was almost as defective as a deletion of an RNase H2 subunit for removing single rNMPs (measured by 2–5 bp deletions in the CAN1 gene), had limited activity in removing R-loops accessible to either RNase H2 or H1, yet displayed remarkably good activity when it interacted with other proteins involved in R-loops associated with DNA replication/repair (Reference 3).
RNase H2 activity is not required in the yeast Saccharomyces cerevisiae, but in mouse, and presumably in humans, Rnaseh2A-, 2B- or 2C-null mutations are lethal, with mouse embryonic development arresting at E8.5. The mouse strain carrying the Rnaseh2a–G37S described above lives longer than the RNaseh2-null mice and retains reduced RNA/DNA hybrid–degradative and RER activities. To examine an RNase H2RED mouse strain, we recently developed mice that are defective in initiating removal of rNMPs in DNA and are examining the effects of this mutation on viability and for other possible defects.
Steric gate in pol eta limits incorporation of rNMPs during DNA replication.
DNA polymerases are able to limit incorporation of ribonucleotides into DNA by sterically limiting binding of the closely related ribonucleotide triphosphates (rNTPs). However, the cellular concentrations of rNTPs are substantially greater than dNTPs, and it is now clear that all three replicative DNA polymerases actually synthesize DNA containing rNMPs. Together with the Woodgate lab (Reference 4), we examined DNA polymerase eta (pol eta), the DNA polymerase important for repair of UV–damaged DNA. Based on other DNA pol steric-gate binding sites, we identified the residue Phe35 as a candidate amino acid limiting incorporation of rNMPs in DNA, for discrimination between rNTPs and dNTPs. Substitution of an Ala for Phe35 opened the active site, allowing incorporation rNMPs. Employing the mutation F35A in pol eta together with the mutated RNase H2RED, we were able to show that rNMPs are incorporated into DNA generating 2–5 bp deletions in short repetitive sequences characteristically found in RNase H2-null mutations. The defect in RNase H2RED permits us to conclude that the mutations are the result of single rNMPs in DNA but not of RNA/DNA hybrids because RNase H2RED retains the ability to hydrolyze RNA of RNA/DNA hybrids. Interestingly, we detected another, new type of mutation in which a deletion of 1 basepair in a run of seven Ts is much more prevalent than the 2–5 bp deletions. In addition, the overall mutation frequency is lower in the pol eta wild-type strain without active RNase H2. We believe that this is a consequence of recognition of single rNMPs in DNA by the mismatch repair pathway, providing another opportunity to repair any mistakes during UV repair by pol eta.
Mitochondrial DNA and RNase H1 in the mouse
During embryonic development, RNase H1 is required for progress beyond day E8.5. A single transcript of the mouse Rnaseh1 gene is translated to make two nearly identical proteins, one localizing to the nucleus and the other to the mitochondrion. We previously showed that the Rnaseh1–deleted embryos fail to amplify mtDNA (mitochondrial DNA), causing developmental arrest. Nuclear DNA replicated normally in Rnase H1–KO embryos. We are examining loss of the Rnaseh1 gene during B-cell development to follow the process of RNase H1 depletion in a simpler system. Following conditional deletion of Rnaseh1 at an early stage of B-cell development in mouse B-cells, we found that resting, naive B-cells are formed but that they are unable to become activated to class-switch to other isotypes (e.g., IgG), and that sera from these mice have a major deficit in antibodies. We are currently determining whether the loss of mitochondrial DNA is the explanation for the inability of the resting B-cells to be completely activated, as well as what changes in mRNA levels are different between the conditional KO and WT Rnaseh1 genes.
J. Brad Holmes, a former graduate student in the NIH-Cambridge graduate student program co-mentored by myself and Ian Holt, examined the roles of RNase H1 in mitochondrial DNA replication by using, among other techniques, analysis of intermediates on two-dimensional gels. Our findings thus far indicate that elevated expression of RNase H1 in mitochondria alters mtDNA replication. In addition, data obtained by Holmes, supporting the existence of RNA/DNA hybrids as replication intermediates, indicate that RNase H1 is important for the removal of RNA primers of mtDNA replication. We concluded that the absence of RNase H1 activity results in accumulation of RNA from the Light Strand Promoter (LSP) fused to DNA, a result that clearly indicates that a transcript from LSP serves to initiate DNA replication of the “H” strand oriH (Reference 5).
- Scientific Director's Intramural Award
- Cerritelli SM, Crouch RJ. Ribonuclease H: the enzymes from eukaryotes. FEBS J 2009; 276:1494-1505.
- Sparks JL, Chon H, Cerritelli SM, Kunkel TA, Johansson E, Crouch RJ, Burgers PM. RNase H2-initiated ribonucleotide excision repair. Mol Cell 2012; 47:980-986.
- Chon H, Spark JL, Rychlik M, Nowotny M, Burgers PM, Crouch RJ, Cerritelli SM. RNase H2 roles in genome integrity revealed by unlinking its activities. Nucleic Acids Res 2013; 41:3130-3143.
- Donigan KA, Cerritelli SM, McDonald JP, Vaisman A, Crouch RJ, Woodgate R. Unlocking the steric gate of DNA polymerase ¿ leads to increased genomic instability in Saccharomyces cerevisiae. DNA Repair 2015; 35:1-12.
- Holmes JB, Akman G, Wood SR, Sakhuja K, Cerritelli SM, Moss C, Bowmaker MR, Jacobs HT, Crouch RJ, Holt IJ. Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci USA 2015; 112:9334-9339.
- Peter Burgers, PhD, Washington University, St. Louis, MO
- Ian Holt, PhD, MRC-Mill Hill, London, United Kingdom
- Herbert C. Morse, MD, Laboratory of Immunopathology, NIAID, Bethesda, MD
- Justin Sparks, PhD, Washington University, St. Louis, MO
- Roger Woodgate, PhD, Laboratory of Genomic Integrity, NICHD, Bethesda, MD
- Nan Yan, PhD, UT Southwestern Medical Center, Dallas, TX