Abstract: Although the basic mechanisms of DNA synthesis are conserved across species, there are differences between simple and complex organisms. In contrast to lower eukaryotes, replication origins in complex eukaryotes lack DNA sequence specificity, can be activated in response to stressful conditions and require poorly conserved factors for replication firing. The response to replication fork damage is monitored by conserved proteins, such as the TIPIN-TIM-CLASPIN complex. The absence of this complex induces severe effects on yeast replication, whereas in higher eukaryotes it is only crucial when the availability of replication origins is limiting. Finally, the dependence of DNA replication on homologous recombination proteins such as RAD51 and the MRE11-RAD50-NBS1 complex is also different; they are dispensable for yeast S-phase but essential for accurate DNA replication in metazoans under unchallenged conditions. The reasons for these differences are not yet understood. Here, we focus on some of these known unknowns of DNA replication.