Cdc45 is an essential protein that together with Mcm2-7 and GINS forms the eukaryotic replicative helicase CMG. Cdc45 seems to be rate limiting for the initial unwinding or firing of replication origins. In line with this view, Cdc45-overexpressing cells fired at least twice as many origins as control cells. However, these cells displayed an about 2-fold diminished fork elongation rate, a pronounced asymmetry of replication fork extension, and an early S phase arrest. This was accompanied by H2AX-phosphorylation and subsequent apoptosis. Unexpectedly, we did not observe increased ATR/Chk1 signaling but rather a mild ATM/Chk2 response. In addition, we detected accumulation of long stretches of single-stranded DNA, a hallmark of replication catastrophe. We conclude that increased origin firing by upregulated Cdc45 caused exhaustion of the single-strand binding protein RPA, which in consequence diminished the ATR/Chk1 response; the subsequently occurring fork breaks led to an ATM/Chk2 mediated phosphorylation of H2AX and eventually to apoptosis.

The mammalian DNA replication program is controlled at two phases, the licensing of potential origins of DNA replication in early gap 1 (G1), and the selective firing of a subset of licenced origins in the synthesis (S) phase. Upon entry into the S phase, serine/threonine-protein kinase ATR (ATR) is required for successful completion of the DNA replication program by limiting unnecessary dormant origin activation. Equally important is its activator, DNA topoisomerase 2-binding protein 1 (TopBP1), which is also required for the initiation of DNA replication after a rise in S-phase kinase levels. However, it is unknown how the ATR activation domain of TopBP1 affects DNA replication dynamics. Using human cells conditionally expressing a TopBP1 mutant deficient for ATR activation, we show that functional TopBP1 is required in suppressing local dormant origin activation. Our results demonstrate a regulatory role for TopBP1 in the local balancing of replication fork firing within the S phase.

Tuberous Sclerosis (TSC) is characterized by exorbitant mTORC1 signalling and manifests as non-malignant, apoptosis-prone neoplasia. Previous reports have shown that TSC-/- cells are highly susceptible to mild, innocuous doses of genotoxic stress, which drive TSC-/- cells into apoptotic death. It has been argued that this hypersensitivity to stress derives from a metabolic/energetic shortfall in TSC-/- cells, but how metabolic dysregulation affects the DNA damage response and cell cycle alterations in TSC-/- cells exposed to genotoxic stress is not understood. We report here the occurrence of futile checkpoint responses and an unusual type of replicative stress (RS) in TSC1-/- fibroblasts exposed to low-dose genotoxins. This RS is characterized by elevated nucleotide incorporation rates despite only modest origin over-firing. Strikingly, an increased propensity for asymmetric fork progression and profuse chromosomal aberrations upon mild DNA damage confirmed that TSC loss indeed proved detrimental to stress adaptation. We conclude that low stress tolerance of TSC-/- cells manifests at the level of DNA replication control, imposing strong negative selection on genomic instability that could in turn detain TSC-mutant tumours benign.

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