Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

Abstract

Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. DNA replication is tightly monitored to ensure that the genome is replicated precisely once per cell cycle and that DNA replication is complete before mitosis begins. Conditions for DNA synthesis are rarely ideal, and a number of obstacles must often be dealt with, such as a damaged DNA template and shortage of deoxynucleoside triphosphates (dNTPs), to allow replication fork progression. Stalling replication forks pose serious threats to genome integrity because they can collapse through disassembly of the replication complex and break (6,11,24). Such damaged forks may subsequently undergo incorrect repair, leading to genetic changes like chromosomal rearrangements (6,24). Recent data have also revealed that activated oncogenes can induce DNA replication stress, defined here as replication-associated DNA damage (2, 3, 10). Oncogene-induced replication stress can lead to additional tumor-promoting genetic changes, but it may also serve as a tumor barrier by activation of cell cycle arrest, apoptosis, and/or senescence during early tumor development (32).WEE1 and CHK1 kinases have major roles in suppressing DNA replication stress (4,23,27,42), and attenuation of their function can contribute to carcinogenesis and cause cell death (40). The massive amount of DNA breakage is likely mediated by DNA endonuclease activity, and recent studies suggest that this is mediated by the endonuclease MUS81 (12,14,15). Notably, the mechanisms by which oncogenes or inhibition of checkpoint kinases can lead to endonuclease-mediated DNA breakage are poorly understood. It is also not fully understood if these breaks also play a role in inducing fork stalling or if they are temporally delayed events secondary to the fork stalling.As both oncogenes and checkpoint kinases are regulators of cyclin-dependent kinase (CDK) activity, we previously proposed that most of the DNA replication str...

A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity

“…In line with our data, a previous study showed that down-regulation of Cdk2, but not Cdk1, rescued the accumulation of γ-H2AX in Wee1-depleted U2OS osteosarcoma cells (35). Mechanistically, elevated Cdk2 activity upon Wee1 inhibition appears to induce S-phaserelated defects, including aberrant origin firing, nucleotide depletion and Mus81-dependent cleavage of stalled replication forks, with γ-H2AX formation as result (35,36). Additionally, Wee1 inhibition was shown to down-regulate ATR and Chk1 in a CDKdependent fashion, which may contribute to the accumulation of γ-H2AX (37).…”

E2F1 and E2F2 prevent replicative stress and subsequent p53-dependent organ involution

“…13,17 However, the cell fates in these cases are different from those observed in pancreatic tissue, as monocyte progenitor cells lacking E2F1/2 result in senescence, and intestinal progenitor cells lacking E2F1-3 lead to p53-independent apoptosis. The mechanism that leads to replication stress in DKO cells is still unknown, and it would be interesting to determine whether it involves illegitimate replication origin firing and fork stalling, as shown for replication stress caused by overexpression of Cdt1 33 or by aberrant CDK activity 34 in some cell types. A controlled CDK activity is key to maintain genome integrity during S phase, 35 and it has been reported that most of the DNA replication stress arises as a result of elevated CDK activity.…”

“…36 Interestingly, this phenotype can be alleviated by nucleotide addition. 34,37 In E2F1 − / − /E2F2 − / − cells, overexpression of replication proteins such as Mcm3, Cdc6 and Orc1, together with the increased Cyclin/CDK activity, could be the source of the observed DNA damage. It remains, however, to be determined what the contributing role of CDK activity in unscheduled DNA replication is, and the role of intracellular nucleotide pools in DKO phenotype.…”

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

“…CDK is limiting for replication initiation, but the pivotal CDK substrates that drive DNA replication were unknown (Beck et al 2012;Jones et al 2013). As phosphorylation of TICRR T969 and S1001 is known to be essential for DNA replication, we asked whether increasing the level of phosphorylated TICRR would be sufficient to stimulate replication initiation and S-phase progression (Boos et al 2011;Kumagai et al 2011).…”