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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
• Kenji Kojima, PhD, Research Collaborator

Damaged DNA is a 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. Two classes of RNases H, Class I and Class II, are present in most organisms.

Human disorders related to RNases H1 and H2. Human patients with mutations in RNASEH1 exhibit a typical mitochondrial muscular phenotype (Reference 1). Our studies were the first to show that RNase H1 is essential for maintenance of mitochondrial DNA. Mice deleted for the Rnaseh1 gene arrest embryonic development at day 8.5 as a result of failure to amplify mitochondrial DNA (Reference 2). Aicardi-Goutières syndrome (AGS), a severe neurological disorder with symptoms appearing at or soon after birth, can be caused by defective human RNase H2 (Reference 3). We are examining mouse models of AGS to gain insight into the human disorder. To understand the mechanisms, functions, substrates, and basic molecular genetics of RNases H, we employ molecular-genetic and biochemical tools in yeast and mouse models.

Contrasts between Class I and Class II RNases H

Many of our investigations over the past few years 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 hetero-trimeric RNase H2 of eukaryotes is similar in its primary amino-acid sequence to the prokaryotic enzyme. RNase H2 can recognize and cleave both RNA/DNA hybrids and a single ribonucleotide (Reference 4) 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).

Several types of RNA/DNA hybrid structures are formed, which are 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 is single or multiple ribonucleotides incorporated into DNA during replication (Reference 4). 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.

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 for 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, one, G37S, with significant loss of RNase H2 activity. Using the 3D structure of the human enzyme that we had determined, we could locate all known mutations in RNase H2 that cause AGS. 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. A mutation near the catalytic center of G37S–mutated RNase H2 found in some AGS patients results in low RNase H2 activity for both embedded ribonucleotides in DNA and RNA/DNA hybrids (Reference 3). We are developing mouse models of AGS to clarify which defects are associated with each RNase H2 activity. Mice bearing the G37S mutation in homozygous form are perinatal lethal, i.e., either dead at birth or die within a few hours of birth (Reference 3).

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 cardiomyopathy, which 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. Viability of these mice is no different than the innate or adaptive-competent mice. Perhaps, there are additional defects in G37S mice that are directly related to viability—not innate immunity. 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 cyclic GMP-AMP synthase (cGAS) 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 cGAS or Sting genes 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 (Reference 3). 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, Hyongi Chon, a former postdoctoral fellow, rationally designed a modified RNase H2 to make an enzyme unable to cleave single ribonucleotides embedded in DNA but that retains RNA/DNA hydrolytic activity. The mutant enzyme, which we call RED (Ribonucleotide Excision Deficient), resolves RNA/DNA hybrids, which are substrates of both RNase H1 and RNase H2. Unlike the mouse and human RNases H2, RNase H2 activity is not required in the yeast Saccharomyces cerevisiae. Taking advantage of the ease with which genetic mutation studies can be conducted in yeast, we found that, in yeast producing the RNase H2^RED enzyme, embedded ribonucleotides (rNMPs) were left in DNA but that the enzyme was potent in removing RNA from RNA/DNA hybrids.

Embryonic lethality of mice Rnaseh2b-KO stains has been attributed to accumulation of rNMPs in DNA, but lethality could result from loss of RNA/DNA hydrolysis or a combination of both rNMP and RNA/DNA hydrolysis defects. To distinguish among the possible causes of embryonic lethality, we generated a mouse that produces the RNase H2^RED enzyme. Mouse embryonic fibroblasts (MEFs) derived from Rnaseh2^RED mice have the same high level of rNMPs as seen in Rnaseh2b-KO MEFs. Interestingly, the Rnaseh2^RED mice also die around the same time as the Rnaseh2b-KO mice. Therefore, lethality of the KO and RED RNase H2 mouse strains lead to embryonic death. We are continuing to analyze these mice and embryos to understand the relationship between early embryonic lethality and the innate immune character of AGS.

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 ribonucleoside triphosphates (rNTPs). However, the cellular concentrations of rNTPs are substantially greater than dNTPs, and it is now clear that all three replicative DNA polymerases do synthesize DNA containing rNMPs. Together with the Woodgate lab, we examined DNA polymerase eta (pol eta), the DNA polymerase important for repair of UV–damaged DNA (Reference 5). Based on other DNA pol steric-gate binding sites, we identified 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 H2^RED, we were able to show that rNMPs are incorporated into DNA generating 2–5 base-pair (bp) deletions in short repetitive sequences characteristically found in RNase H2–null mutations. The defect in RNase H2^RED permits us to conclude that the mutations are the result of single rNMPs in DNA but not of RNA/DNA hybrids, because RNase H2^RED retains the ability to hydrolyze RNA of RNA/DNA hybrids. Interestingly, we detected another, new type of mutation in which a deletion of one base pair in a run of seven thymines 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 (References 1 and 2). 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, whereas nuclear DNA replicated normally in Rnaseh1–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 such 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 differ 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, two-dimensional gel analysis of intermediates. 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 origin of replication (oriH) (Reference 2).

Replication and transcription of mitochondrial DNA are intimately associated with a control region, a displacement D-loop. The loop has a short 7S DNA that is hydrogen-bonded to one strand, thereby displacing the third strand in this region. We recently reported that there is an R-loop complementary to the other (displaced) DNA strand of the control region (Reference 1). The D- and R-loops are independent, i.e., there are few if any four-stranded molecules. The control-region R-loops are potential substrates for RNase H1. In cells with a pathological mutation in RNASEH1, R-loops are less abundant in the control region, and mitochondrial DNA is aggregated. The findings implicate RNA and RNase H1 in the physical separation of mitochondrial DNA.

Publications

1. Akmana G, Desaia R, Bailey LJ, Yasukawab T, Rosa ID, Durigon R, Holmes JB, Moss CF, Mennuni M, Houlden H, Crouch RJ, Hanna MG, Pitceathly RDS, Spinazzola A, Holt IJ. Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria. Proc Natl Acad Sci USA 2016;113:E4276-E4285.
2. 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.
3. Pokatayev V, Hasin N, Chon H, Cerritelli SM, Yan N, Crouch RJ. RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS-STING innate immune sensing pathway in mice. J Exp Med 2016;213:329-336.
4. Cerritelli SM, Crouch RJ. The balancing act of ribonucleotides in DNA. Trends Biochem Sci 2016;41:434–445.
5. Donigan KA, Cerritelli SM, McDonald JP, Vaisman A, Crouch RJ, Woodgate R. Unlocking the steric gate of DNA polymerase eta leads to increased genomic instability in Saccharomyces cerevisiae. DNA Repair 2015;35:1-12.

Collaborators

• Patricia J. Gearhart, PhD, Laboratory of Molecular Biology and Immunology, NIA, Baltimore, MD
• Ian Holt, PhD, MRC-Mill Hill, London, United Kingdom
• Herbert C. Morse, MD, Laboratory of Immunopathology, NIAID, Bethesda, MD
• Roger Woodgate, PhD, Laboratory of Genomic Integrity, NICHD, Bethesda, MD
• Nan Yan, PhD, UT Southwestern Medical Center, Dallas, TX

Contact

For more information, email crouch@helix.nih.gov or visit http://sfr.nichd.nih.gov.

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