Explanation Research in the Section on Formation of RNA is directed toward understanding the interaction between RNA and DNA that perturb the DNA and how the cell responds to these RNA-containing DNAs. The AIDS virus, HIV, employs RNA as its genome and when copied into DNA RNA/DNA hybrids are intermediates that require RNase H activity, an enzyme that removes the RNA after it is copied by the viral DNA polymerase (Reverse Transcriptase or RT). RNase H is an essential part of RT and could be a target for therapeutic drugs. RNA/DNA hybrids are also present in normal cells, occasionally forming during transcription producing R-loops in which the RNA displaces one strand of DNA and forms a duplex with the complementary DNA strand. In addition, recombination proteins can form RNA/DNA hybrids by displacing one DNA strand while annealing complementary RNA to the other DNA strand. If unresolved, these R-loops lead to genome instability. The endogenous RNases H usually remove these R-loops. Aicardi Goutieres Syndrome (AGS) is a rare autoimmune disorder with severe neurological problems that can be caused by defects in human RNase H2. AGS mimics in utero viral infection including loss of white matter in the brain and producing high levels of interferon alpha in the cerebral spinal fluid. Mammalian RNases H2 can degrade RNA/DNA hybrids but can also recognize a single ribonucleotide in duplex DNA and initiates its removal. Current proposals suggest it is the failure to remove the missincorporated ribonucleotides results in DNA damage causing AGS. However, both RNA/DNA and single ribonucleotides in DNA remain when RNase H2 is not present; conditions tested so far. Thus, the jury is still out on the relationship between RNase H2 defects and AGS. We have been employed Saccharomyces cerevisiae as a model organism to examine conditions under which the two activities of RNase H2 are required. Interestingly, we have example where either RNase H1 or H2 can resolve the same R-loops and another in which R-loops are only degraded by RNase H2. To gain more insight into the effects of the same into AGS-related mutations in mammals, we have generated a mouse which expresses a mutant form of RNase H2 seen in a few AGS patients and have been examining the properties of the mouse and mouse tissues. The mice homozygous for the mutation are born dead or or die soon after birth. One model for defects in RNase H2 (and other proteins that when defective cause AGS) is activation of endogenous retroviruses which can result in induction of an innate immune response ultimately leading to an auto immune condition. While our results are still in an incomplete stage, we find no evidence of autoimmunity. RNase H1 is present in mitochondria (mt) and nuclei of mammalian cells and is required during embryonic development to generate mtDNA. Its function in the nucleus is not completely understood but it too is likely important for resolving R-loops with partial overlapping action with RNase H2. It has been shown that R-loop formation at repetitive sequences can lead to recombination that result in expansion (duplication) or deletion. Several human disorders arise when a triplet repeat such as CAG glutamine (Q) codon expand producing proteins containing long stretches of one amino acid (polyQ for CAG expansion). Often these proteins become aggregated and can affect the cell due to these complexes. In some cases, the expansion has direct affects on the function of the protein with the expansion. It is easy to imagine that the displaced DNA strand of R-loops can reanneal to its complementary DNA strand in multiple ways leading to repair and expansion or contraction. We are examining ataxic mice that arose in our RNase H1 knockout strain may have such triplet expansions. The role of RNase H1 in mtDNA replication is beginning to become clear from some of the studies carried out in collaboration with Ian Holt at the National Institute for Medical Research, MRC London. These advances have been made in part by using mouse embryo fibroblasts with defects in RNase H1 production that lead to cell death and accumulation of RNA/DNA in mtDNA. The model developed in Holts lab suggests RNA is a major intermediate in mtDNA replication, possibly by a mechanism similar to that described for RNA/DNA hybrid formation mediated by a recombination-related protein.

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45
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2016
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U.S. National Inst/Child Hlth/Human Dev
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Baba, Misato; Kojima, Kenji; Nakase, Rihoko et al. (2017) Effects of neutral salts and pH on the activity and stability of human RNase H2. J Biochem :
Malfatti, Matilde Clarissa; Balachander, Sathya; Antoniali, Giulia et al. (2017) Abasic and oxidized ribonucleotides embedded in DNA are processed by human APE1 and not by RNase H2. Nucleic Acids Res 45:11193-11212
Maul, Robert W; Chon, Hyongi; Sakhuja, Kiran et al. (2017) R-Loop Depletion by Over-expressed RNase H1 in Mouse B Cells Increases Activation-Induced Deaminase Access to the Transcribed Strand without Altering Frequency of Isotype Switching. J Mol Biol 429:3255-3263
Akman, Gokhan; Desai, Radha; Bailey, Laura J et al. (2016) Pathological ribonuclease H1 causes R-loop depletion and aberrant DNA segregation in mitochondria. Proc Natl Acad Sci U S A 113:E4276-85
Pokatayev, Vladislav; Hasin, Naushaba; Chon, Hyongi et al. (2016) RNase H2 catalytic core Aicardi-Goutières syndrome-related mutant invokes cGAS-STING innate immune-sensing pathway in mice. J Exp Med 213:329-36
Cerritelli, Susana M; Crouch, Robert J (2016) The Balancing Act of Ribonucleotides in DNA. Trends Biochem Sci 41:434-445
Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R et al. (2015) Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication. Proc Natl Acad Sci U S A 112:9334-9
Gaidamakov, Sergei; Maximova, Olga A; Chon, Hyongi et al. (2014) Targeted deletion of the gene encoding the La autoantigen (Sjögren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss. Mol Cell Biol 34:123-31
Chon, Hyongi; Sparks, Justin L; Rychlik, Monika et al. (2013) RNase H2 roles in genome integrity revealed by unlinking its activities. Nucleic Acids Res 41:3130-43
Sparks, Justin L; Chon, Hyongi; Cerritelli, Susana M et al. (2012) RNase H2-initiated ribonucleotide excision repair. Mol Cell 47:980-6

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