Ribonucleases (RNases) H1 and H2 are endonucleases that hydrolyze the RNA strand of RNA-DNA hybrids forming at the chromosomal level as well as extra-chromosomal hybrids. Extra-chromosomal RNA-DNA hybrids can frequently occur in cells as intermediate structures in the process of reverse transcription and generation of cDNA by retrotransposition. It is known that mutations in RNase H2 are found in Aicardi-Goutières syndrome (AGS) patients. AGS is a rare but severe immune-mediated neurodevelopmental disorder. Currently, the mechanism by which defects in RNase H2 cause AGS is still unclear. We hypothesized that defects in RNases H, including those associated with AGS can trigger the accumulation of extra-chromosomal RNA-DNA hybrids. Thus, we speculate that increased stability of such free RNA-DNA hybrid structures could be a likely trigger for stimulating the autoimmune system, mimicking a viral infection in AGS patients. RNase H2 protein subunits of human and yeast Saccharomyces cerevisiae RNase H2 proteins have conserved amino acid sequences. Based on the similarity between human and yeast RNase H2, we thought to utilize S. cerevisiae as a research model to generate and study several AGS-related mutants. Initially, we set up an assay to detect retrotransposition activity in the budding yeast by introducing a recombinant DNA which includes a Ty1 retrotransposable element fused to an inactive his3 marker gene. To test whether the retrotransposition assay works in our yeast strains, we treated yeast cells with phosphonoformic acid (PFA) or knocked out DBR1 gene coding for the RNA lariat debranching enzyme. Both approaches strongly reduced the frequency of retrotransposition in our strains, demonstrating that the system was working as expected. Next, we examined whether yeast cells with defective forms of RNases H or AGS-orthologous mutants of RNase H2 had altered retrotransposition activity compared with cells with wild-type RNases H. Results showed that the retrotransposition activity was repressed in the absence of both types of RNase H. In addition, AGS-related mutants showed decreased retrotransposition frequencies when RNase H1 was also knocked-out. These findings are relevant to uncover the mechanism of the AGS.