Cancer is a disease associated with genomic instability that often results from oncogene activation. This in turn leads to hyperproliferation and replication stress. However, the molecular mechanisms that underlie oncogene-induced replication stress are still poorly understood. Oncogenes such as HRASV12 promote proliferation by upregulating general transcription factors to stimulate RNA synthesis. Here we investigate whether this increase in transcription underlies oncogene-induced replication stress. We show that in cells overexpressing HRASV12, elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which together with R-loop accumulation results in replication fork slowing and DNA damage. Furthermore, overexpression of TBP alone causes the hallmarks of oncogene-induced replication stress, including replication fork slowing, DNA damage and senescence. Consequently, we reveal that increased transcription can be a mechanism of oncogene-induced DNA damage, providing a molecular link between upregulation of the transcription machinery and genomic instability in cancer.

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 ( Keywords: Cyclin E; replication fork; origin firing; DNA damage; homologous recombination INTRODUCTION Faithful replication of the genome is essential to maintain genomic stability and prevent cancer-promoting mutations. If the progression of replication forks is impaired, this can lead to replicationassociated DNA damage, also known as replication stress. It is now recognized that replication stress induced by oncogenes is an important factor that underlies the activation of the ataxia telangiectasia mutated (ATM)-and p53-mediated tumor barriers of senescence and apoptosis. [1][2][3][4][5][6][7] There is currently no coherent model to explain how oncogenes induce replication stress and DNA damage. Oncogenes of the growth factor signaling pathways generally activate cell proliferation by deregulating the transition from G1 to S phase of the cell cycle.8 Deregulated S-phase entry leading to aberrant DNA replication is therefore likely a principal mechanism of oncogene-induced replication stress. It has been reported that overexpression of the oncogenes HPV-16 E6/E7 and Cyclin E leads to S-phase entry in the presence of insufficient nucleotide pools, leading to impaired DNA replication fork progression and DNA damage.9 However, the underlying mechanism for the nucleotide depletion observed in the presence of oncogenes is not clear, and not all oncogenes that deregulate S-phase entry induce DNA damage. 10 The mechanisms of aberrant DNA replication and oncogene-induced replication stress therefore deserve further scrutiny.During a normal cell cycle, not only entry into S phase but also the timing of DNA replication initiation during S phase is carefully regulated by cell cycle and checkpoint signaling pathways.

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.

We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a “backup” for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.

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