The genetic information (i.e., genome) of a human cell is encoded in strands of DNA that assemble into an antiparallel DNA double helix. Each time a cell divides, the genome must be faithfully replicated and transferred to a daughter cell for genetic inheritance. The former occurs during S-phase of the cell cycle and relies on high-fidelity, i.e. “replicative,” DNA polymerases that read template DNA strands and synthesize their complementary DNA. Additional “core” proteins and enzymes are also involved and the basic mechanism of human DNA replication has been deciphered. However, it is currently unknown how DNA replication is achieved on genomic DNA within a human cell. For example, the majority of proteins and enzymes implicated in human DNA replication are dynamically modified by chemical and protein moieties in vivo. Currently, the functional role and regulation of many of these modifications is unknown. Furthermore, genomic DNA is continuously subjected to damage from reactive metabolites and environmental mutagens. Prominent examples are modifications (lesions) to the native template DNA bases that alter or eliminate their base pairing capability. It is unclear how DNA lesions are accommodated during S-phase without compromising the fidelity of DNA replication. We aspire to decipher how efficient and faithful replication of the human genome is achieved within the highly-complex, dynamic, and reactive cellular environment. To do so, we employ a multi-disciplinary, collaborative approach, combining biophysical, biochemical, and molecular and cellular biology techniques to; 1) identify cellular factors involved in various aspects of human DNA replication and; 2) re-constitute human DNA replication in various biological scenarios and at various levels of complexity.