Topoisomerase III beta (TOP3B) is a conserved type IA nucleic acid processing enzyme that catalyses RNA strand passage reactions. Together with partner proteins, TOP3B is implicated in the resolution of R-loops that are generated during transcription. Previous studies in knockout mice show a slight reduction in lifespan, chromosomal defects in germ cells and progressive infertility across generations. Furthermore, mouse embryonic fibroblasts are sensitive to DNA-damaging agents and have impaired up-regulation of p53. To further understand whether the loss of TOP3B increases genome instability events we have generated knockout human cell lines. We show elevated rates of genome instability in the form of micronuclei, gH2Ax signals and chromosome segregation defects. We are additionally investigating a family with a homozygous deletion of the TOP3B gene with overlapping phenotypes observed in cell line and animal model systems.