Accurate and complete genome replication is essential for all life. Deregulation of DNA replication can lead to genome instability and is linked to several human disorders. My research focuses on the cellular regulation of DNA replication in diverse eukaryotic and prokaryotic cells. Eukaryotic genomes are replicated in a reproducible temporal order. To investigate this, we have developed high-throughput sequencing methods to quantitatively measure at high resolution the temporal order of genome replication. This approach is driving fundamental discoveries about how genomes replicate across the domains of life. For example, comparing genome replication in different species allowed us to discover that replication origin activity is regulated in cis and that these mechanisms contribute to faithful chromosome inheritance. Recently, we have started to investigate the physiological significance of replication timing. We have identified genomic features that show an evolutionary conservation in their replication time, for example, genes that replicate early in S phase across many species. To test the physiological importance, we specifically delayed the replication time of such genes and discovered that this resulted in reduced gene expression. This is one of the first demonstrations of a physiological requirements for regulated replication time and provides a direct link between replication time and gene expression.