DNA fulfills two critical roles in a cell by being the template for both DNA replication and RNA transcription. During evolution, eukaryotic cells have adopted multiple strategies to coordinate these two processes to prevent or minimize conflicts that might arise between them. Here, we review the strategies employed by cells to simultaneously accommodate the machineries carrying out DNA replication and transcription, and the mechanisms that are brought into play for resolving conflicts when they do arise. We focus on a group of the so-called ‘difficult-to-replicate’ loci in the human genome, which include chromosome fragile sites, the ribosomal DNA, and telomeres. A failure to resolve conflicts arising between replication and transcription can lead to genome instability, as well as to the initiation of cancer and other age-associated diseases. Understanding the mechanisms required for the resolution of these conflicts could, therefore, open up new therapeutic avenues.