Accurately copying its whole genome is perhaps the most important task of the cell. Mistakes in DNA replication can result in cell death or, potentially worse, in mutagenesis and genomic instability, which in turn can lead to uncontrolled proliferation, the basis of cancer. Elucidating the factors involved in DNA replication and understanding the mechanisms by which human cells guarantee the precise duplication of billions of base pairs and thereby appropriate proliferation are of utmost importance when aiming at defeating several diseases, among which cancer stands out as one of the leading causes of death in the world. While the actual enzymatic activity of DNA synthesis is accomplished by DNA polymerases, a remarkable set of extra factors is required for replication within the cell. Though the core replication components seem to be conserved from yeast to humans, in mammals the putative homologues of several key factors likely act with different mechanisms or have additional functions, as suggested by the low homology level and presence of extra domains. Thus, the object of my proposal is to identify novel factors involved in normal DNA replication progression and the response to replicative stress at the replisome level, specifically in human cells. I will put additional emphasis on elucidating the function of known metazoan-specific factors, which likely play a key role in the complex regulatory mechanisms required in higher organisms. To this end, I will combine various cutting edge methods, like nanobody-based sequential purifications, SILAC mass spectrometry and 3D-SIM microscopy, to develop assays to isolate active replisomes from human cells and study the identified factors mechanistically. This strategy will allow me to exploit and significantly expand my technical expertise and, complemented by the selected world-class host scientist and institute, to grow into an independent scientist and enhance Europe’s research excellence.