Ub has an essential role within the DNA double strand break (DSB) response which is well documented. However the role of ubiquitin (Ub) in the regulation of replication is an emerging area of research. This thesis investigates how two deubiquitinating enzymes (DUBs), POH1 and USP50, regulate DSB repair and replication respectively. A screen of 103 siRNAs against putative DUBs in the human genome, measuring the amount of conjugated Ub after release from HU-induced damage, identified the proteasome associated DUB, POH1 as being important in regulating Ub-conjuagtes after damage. Further work found that POH1 restricts the K63-linked Ub at DSBs and consequently 53BP1 foci formation. This appears to regulate repair of breaks by Non-homologous end-joining (NHEJ). The DUB screen also identified USP50 as having significantly reduced levels of conjugated Ub after damage. USP50 is an inactive DUB with Ub-binding activities, which has a role in preventing formation of Mus81-dependent DSBs during replication, with depletion sensitising cells to replication-stress. Therefore this works demonstrates a role for USP50 in genomic stability during replication. In this thesis I demonstrate the role of these two DUBs in DSB repair and replication respectively, providing potential therapeutic targets.