Project Summary Genome integrity depends on a robust DNA replication program and the activity of replication-coupled repair pathways that operate during different phases of the cell cycle. My laboratory has had a longstanding interest in the causes and consequences of replication stress. Replication stress arises when lesions in the genome persist due to repair deficiencies or when components of the replication machinery are dysfunctional. Although disease- causing mutations in essential replication factors are rare, they can cause pleiotropic and severe disorders, such as immunodeficiency, cardiomyopathy, or growth defects. In recent years, we have investigated the molecular mechanism that underlies these rare diseases. We have identified compound heterozygous patient mutations in the replication factor minichromosome maintenance protein 10 (MCM10), and have modeled them in human somatic cell lines. Although these mutations cause relatively mild cellular replication defects, they pose significant problems to telomere maintenance. One caveat of the current cell models is that they are immortalized and express telomerase constitutively. To better understand the impact of replication defects in the context of cellular development of affected tissues, we propose to engineer genome-edited induced pluripotent stem cells and differentiate them into specific cell types in vitro. This presents a valuable alternative to animal models which, relevantly, do not fully mimic telomere homeostasis in humans. Moreover, we are interested in the pathways that cells activate for survival under conditions of mild replication stress. Previous work has identified a network based on ubiquitination and SUMOylation, and ring finger protein 4 (RNF4) as a key component. RNF4 is a SUMO- targeted E3 ubiquitin ligase that has been implicated in double-strand break repair, however, its role at replication forks and in telomere maintenance is not well understood. A genetic interaction screen has identified Bloom helicase (BLM), a RecQ-family helicase that causes premature aging, and ubiquitin specific peptidase 7 (USP7), a deubiquitinase, as strong negative interactors. Mutations in USP7 have been linked to rare neurodevelopmental disorders, but its cellular action has remained obscure. Interestingly, USP7 and BLM also regulate DNA replication and telomere length. We will investigate the relationship between RNF4, USP7 and BLM in chromosome inheritance in telomerase-positive and -negative cells. Lastly, a common feature of replication stress is under-replication due to an inability to duplicate the entire genome. As a result, single- stranded gaps persist that can either be filled by post-replicative repair that is regulated by the ubiquitination of PCNA or, as a last resort, by mitotic DNA synthesis (MiDAS). MiDAS is a break-induced replication (BIR)-like pathway that, unlike a classical replication fork, copies DNA by displacement synthesis. We will study how ubiquitinated PC...