It has become increasingly clear that oncogenes not only provide aberrant growth signals to cells but also cause DNA damage at replication forks (replication stress), which activate the ataxia telangiectasia mutated (ATM)/p53-dependent tumor barrier. Here we studied underlying mechanisms of oncogene-induced replication stress in cells overexpressing the oncogene Cyclin E. Cyclin E overexpression is associated with increased firing of replication origins, impaired replication fork progression and DNA damage that activates RAD51-mediated recombination. By inhibiting replication initiation factors, we show that Cyclin E-induced replication slowing and DNA damage is a consequence of excessive origin firing. A significant amount of Cyclin E-induced replication slowing is due to interference between replication and transcription, which also underlies the activation of homologous recombination. Our data suggest that Cyclin E-induced replication stress is caused by deregulation of replication initiation and increased interference between replication and transcription, which results in impaired replication fork progression and DNA damage triggering the tumor barrier or cancer-promoting mutations.Oncogene advance online publication, 3 September 2012; doi:10.1038/onc.2012.387.