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...

Lens epithelium-derived growth factor p75 splice variant (LEDGF) is a chromatin-binding protein known for its antiapoptotic activity and ability to direct human immunodeficiency virus into active transcription units. Here we show that LEDGF promotes the repair of DNA double-strand breaks (DSBs) by the homologous recombination repair pathway. Depletion of LEDGF impairs the recruitment of C-terminal binding protein interacting protein (CtIP) to DNA DSBs and the subsequent CtIP-dependent DNA-end resection. LEDGF is constitutively associated with chromatin through its Pro-Trp-Trp-Pro (PWWP) domain that binds preferentially to epigenetic methyl-lysine histone markers characteristic of active transcription units. LEDGF binds CtIP in a DNA damage-dependent manner, thereby enhancing its tethering to the active chromatin and facilitating its access to DNA DSBs. These data highlight the role of PWWP-domain proteins in DNA repair and provide a molecular explanation for the antiapoptotic and cancer cell survival-activities of LEDGF.

WEE1 and CHK1 jointly regulate Cdk activity to prevent DNA damage during replication.

The molecular events occurring following the disruption of DNA replication forks are poorly characterized, despite extensive use of replication inhibitors such as hydroxyurea in the treatment of malignancies. Here, we identify a key role for the FBH1 helicase in mediating DNA double-strand break formation following replication inhibition. We show that FBH1-deficient cells are resistant to killing by hydroxyurea, and exhibit impaired activation of the pro-apoptotic factor p53, consistent with decreased DNA double-strand break formation. Similar findings were obtained in murine ES cells carrying disrupted alleles of Fbh1. We also show that FBH1 through its helicase activity co-operates with the MUS81 nuclease in promoting the endonucleolytic DNA cleavage following prolonged replication stress. Accordingly, MUS81 and EME1-depleted cells show increased resistance to the cytotoxic effects of replication stress. Our data suggest that FBH1 helicase activity is required to eliminate cells with excessive replication stress through the generation of MUS81-induced DNA doublestrand breaks.

No abstract