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. If unresolved, these R-loops can 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 an RNase H2 seen in a few AGS patients and have been examining the properties of the mouse and mouse tissues. 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 mice that 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 of the Mitochondrial Unit at the MRC Cambridge. 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. Mouse RNase H1 is translated from a single messenger RNA to produce the same protein to reside in nuclei and in mitochondria. Production of the two forms depends on a translation process that we have found to be conserved in many eukaryotic organisms and in other DNA transacting proteins targeted to both the nucleus and mitochondria.
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