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 Goutires 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 incorporated 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. To address this question, we have employed Saccharomyces cerevisiae as a model organism to examine conditions under which the two activities of RNase H2 are required. We call this enzyme RNase H2RED (RNA Excision Defective). Interestingly, we have an 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, RNase H2G37S, and a mouse expressing only the RNase H2RED protein. We have been examining the properties of the mouse and mouse tissues. The mice homozygous for the Rnaseh2G37S mutation are born dead or die soon after birth. DNA damage leads to induction of the cGAS/Sting innate immune response pathway. However, loss of an innate response by genetically inactivating that pathway does not permit formation of viable pups. We have found mice expressing only the RNase H2RED protein are as defective as mice with no RNase H2, where embryonic development is defective as early as E9.5. This finding is consistent with failure to remove ribonucleotides (rNMPs) in DNA causes early embryonic lethality. Interestingly, mice that are heterozygous (Rnaseh2RED/G37S) are also early embryonic lethal. Our results indicate the two mutant forms on RNase H2RED competes with RNase H2G37S allowing retention of more rNMPs in DNA. This is confirmed by the numbers of rNMPs in DNA for the heterozygous mice. The mice with only RNase H2G37S contain more rNMPs than normal mice but still progress development to birth. DNA damage in RNase H2RED mice activates p53 response whereas such a response is not observed in RNase H2G37S mice. This leads us to conclude that there is a threshold of rNMPs required to activate the p53-dependent pathway which is between the number in the RED and G37S mice. In collaboration with Dr. Aziz El Hage, Dr. Susana M. Cerritelli has used S. cerevisiae to modulate the number of rNMPs in DNA by studying the regulation of ribonucleotide reductase (RNR). The ratio of rNTPs/dNTPs is a major contributor to the number of rNMPs incorporated into DNA during replication. RNR can be genetically manipulated to be more and less active. Cerritelli and El Hage have altered many genes controlling RNR and have confirmed the RNR interactive-pathway as well as found important new findings in how RNase H2 respond to these change in RNR. These findings about abundance of rNMPs in DNA are major contributions in defining the incorporation of rNMPs into DNA and the consequences of failure to remove them in both mouse and yeast models of properties of the human disorder, Aicardi-Goutires Syndrome.
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