Increased replication initiation and conflicts with transcription underlie Cyclin E-induced replication stress

Abstract

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.

Deregulated origin licensing leads to chromosomal breaks by rereplication of a gapped DNA template

“…S6B,C). The latter two features closely resemble the effects of oncogene activation (Neelsen et al 2013) and thus most likely reflect the licensing defects common to these genetic conditions (Hook et al 2007;Blow and Gillespie 2008) and their consequences in terms of nucleotide depletion (Bester et al 2011) and/or interference with transcription (Jones et al 2013). Although ssDNA sensors (e.g., RPA chromatin loading and PCNA ubiquitylation) ( Fig.…”

Oncogenes create a unique landscape of fragile sites

“…In early stages of cancer development, oncogene activation leads to replication stress [1][2][3][4] . Mechanisms by which oncogenes can induce replication stress include insufficient nucleotide pools to support extensive replication 4,5 , lack of other replication factors 6 and collision between replication and transcription 7 . In addition, a recent study showed that loss of FHIT expression encoded by the fragile histidine triad gene located in the fragile site locus FRA3B can initiate replication stress and continue genomic instability 8 .…”

Replication stress and cancer: It takes two to tango

“…The mutation of MCM4 or MCM2 reduces the activity of the MCM complex, limiting the amount of available back-up origins, which results in increased RS, genomic instability and a cancer-prone phenotype [14][15][16]. In contrast, the overexpression of certain oncogenes (Myc, Ras…) has the opposite effect and increases the firing of origins of replication, leading to a depletion of the cellular pool of nucleotides (dNTPs) [17][18][19][20][21]. The reduced level of dNTPs slows down the progression of the forks and increases the chance of fork stalling per se.…”

“…Moreover, overexpression of RNR, rather than its loss, is frequently found in human cancers [22]. Regardless of the mechanism, supplementation with nucleosides has been shown to limit oncogene-induced RS in vitro, Besides any effect on the nucleotide pool, the higher number of active origins generates more replication-transcription collisions that lead to stalled replication forks [18], expression of common fragile sites and genome instability [24]. There is increasing evidence showing that the formation of RNA-DNA hybrid called R-loops is central to the replicationtranscription collisions [25].…”