Aicardi-Goutières syndrome (AGS) is an autosomal recessive encephalopathy with low incidence. The disease is caused by mutations in the genes encoding for TREX1, SAMHD1, ADAR, IFIH1 and the three genes encoding for the heterotrimeric RNase H2 enzyme. Biallelic mutations in any of the genes cause elevated type I interferon levels in the cerebrospinal fluid (CSF), the hallmark of AGS. In AGS patients, increased type I interferon levels cause massive inflammation in the brain that leads to mental and physical retardation that likely cause death in early childhood. AGS shows significant overlap with the prototypic autoimmune disease systemic lupus erythematosus (SLE). Like AGS patients, SLE patients are also characterized by increased type I interferon levels, anti-nuclear autoantibodies (ANAs) and arthritis. Moreover, heterozygous mutations in TREX1, SAMHD1 and RNase H2 are also found in a small fraction of SLE patients. Due to the genetic, molecular and clinical overlap, AGS is regarded as a monogenic variant of SLE. This overlap allows for the investigation of SLE pathomechanisms using genetically engineered mouse models with AGS-related mutations. In order to generate a mouse model that allows for the identification of pathomechanisms in AGS patients with mutations in the genes encoding for the RNase H2 enzyme, we generated mice with deficiency for the RNase H2 enzyme. Mice with complete deficiency for the RNase H2 enzyme (Rnaseh2c-/- or Rnaseh2bKOF/KOF) died perinatally or were stillborn. Mouse embryonic fibroblasts (MEFs) from E14.5 Rnaseh2bKOF/KOF embryos displayed impaired proliferation that was caused by the accumulation of MEF cells in G2/M of the cell cycle which increased with cultivation time or if MEF cells were isolated from E18.5 Rnaseh2bKOF/KOF embryos. Gene expression analysis of E14.5 fetal liver cells revealed a robust p53-mediated DNA damage response with the cell cycle inhibitor cyclin- dependent kinase inhibitor 1a (Cdkn1a, p21) being the most up-regulated gene. We found increased numbers of phosphorylated histone H2AX (γH2AX) in fetal liver and thymus cells from E18.5 Rnaseh2bKOF/KOF embryos, indicative of DNA double-strand breaks. Finally, we could show increased ribonucleotide loads in genomic DNA from embryos that were completely deficient for the RNase H2 enzyme. Collectively, we have demonstrated that complete RNase H2 deficiency causes persistent genomic ribonucleotide loads that render the DNA instable and prone to DNA strand breaks. DNA damage leads to the activation of p53 that in turn activates the cell cycle inhibitor p21 that inhibits cell cycle progression and likely causes accumulation of RNase H2-deficient cells in G2/M. To bypass early lethality we also generated bone marrow chimera and cell type-specific knockouts of the Rnaseh2b gene. While fetal liver cells of E14.5 Rnaseh2bKOF/KOF embryos could maintain hematopoiesis of irradiated recipient mice for almost one year, bone marrow cells from these primary recipients failed to reconstitute lethally irradiated mice in a secondary transfer. In line with this observation, VavCre-mediated deletion of the Rnaseh2b gene caused a more than hundred fold reduction of peripheral blood B cells, while B cell numbers remained unaltered upon CD19Cre-mediated deletion that occurs much later in B cell development. These data suggested that RNase H2 deficiency leads to the accumulation of genomic ribonucleotides that might cause the accumulation of a so far uncharacterized DNA damage species with increasing cell cycle passages. The data further supported our hypothesis that the impact of RNase H2 deficiency is determined by the number of cell proliferation. Finally, an epidermis-specific knockout of the Rnaseh2b gene displayed the most dramatic phenotype. Knockout mice were characterized by hyperpigmentation, hair loss and spontaneous ulcerations of the skin. Microscopically, these mice displayed moderate thickening of the epidermis and dermal fibrosis as indicated by increased collagen deposition. Macroscopic skin phenotypes were completely dependent on p53 expression, since concomitant deletion of the p53 gene rescued mice from hyperpigmentation, hair loss and ulcerations. This data demonstrated that Rnaseh2b deficiency in the epidermis may also lead to DNA damage and subsequent p53 activation as shown for fetal liver from E14.5 RNase H2-deficient embryos. Preliminary data also indicate an increased incidence of cancer formation in RNase H2/p53 double knockouts, identifying the RNase H2 enzyme as an important tumor suppressor.