DNA replication is a fundamental process of the cell that ensures accurate duplication of the genetic information and subsequent transfer to daughter cells. Various pertubations, originating from endogenous or exogenous sources, can interfere with proper progression and completion of the replication process, thus threatening genome integrity. Coordinated regulation of replication and the DNA damage response is therefore fundamental to counteract these challenges and ensure accurate synthesis of the genetic material under conditions of replication stress. In this review, we summarize the main sources of replication stress and the DNA damage signaling pathways that are activated in order to preserve genome integrity during DNA replication. We also discuss the association of replication stress and DNA damage in human disease and future perspectives in the field.

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.

Oncogenic stimuli trigger the DNA damage response (DDR) and induction of the alternative reading frame (ARF) tumor suppressor, both of which can activate the p53 pathway and provide intrinsic barriers to tumor progression. However, the respective timeframes and signal thresholds for ARF induction and DDR activation during tumorigenesis remain elusive. Here, these issues were addressed by analyses of mouse models of urinary bladder, colon, pancreatic and skin premalignant and malignant lesions. Consistently, ARF expression occurred at a later stage of tumor progression than activation of the DDR or p16INK4A , a tumor-suppressor gene overlapping with ARF. Analogous results were obtained in several human clinical settings, including early and progressive lesions of the urinary bladder, head and neck, skin and pancreas. Mechanistic analyses of epithelial and fibroblast cell models exposed to various oncogenes showed that the delayed upregulation of ARF reflected a requirement for a higher, transcriptionally based threshold of oncogenic stress, elicited by at least two oncogenic 'hits', compared with lower activation threshold for DDR. We propose that relative to DDR activation, ARF provides a complementary and delayed barrier to tumor development, responding to more robust stimuli of escalating oncogenic overload.

Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network. Surprisingly, induction of p73, an established E2F1 target, was also DNA damage response-dependent. Furthermore, a global proteome analysis associated with bioinformatics revealed novel E2F1-regulated genes and potential E2F1-driven signaling networks that could provide useful targets in challenging this aggressive neoplasm by innovative therapies.

Unresolved replication intermediates can block the progression of replication forks and become converted into DNA lesions, hence exacerbating genomic instability. The p53-binding protein 1 (53BP1) forms nuclear bodies at sites of unrepaired DNA lesions to shield these regions against erosion, in a manner dependent on the DNA damage kinase ATM. The molecular mechanism by which ATM is activated upon replicative stress to localize the 53BP1 protection complex is unknown. Here we show that the ATM-INteracting protein ATMIN (also known as ASCIZ) is partially required for 53BP1 localization upon replicative stress. Additionally, we demonstrate that ATM activation is impaired in cells lacking ATMIN and we define that ATMIN is required for initiating ATM signaling following replicative stress. Furthermore, loss of ATMIN leads to chromosomal segregation defects. Together these data reveal that chromatin integrity depends on ATMIN upon exposure to replication-induced stress.

Defects in DNA repair can cause various genetic diseases with severe pathological phenotypes. Fanconi anemia (FA) is a rare disease characterized by bone marrow failure, developmental abnormalities, and increased cancer risk that is caused by defective repair of DNA interstrand crosslinks (ICLs). Here, we identify the deubiquitylating enzyme USP48 as synthetic viable for FA-gene deficiencies by performing genome-wide loss-of-function screens across a panel of human haploid isogenic FA-defective cells (FANCA, FANCC, FANCG, FANCI, FANCD2). Thus, as compared to FA-defective cells alone, FA-deficient cells additionally lacking USP48 are less sensitive to genotoxic stress induced by ICL agents and display enhanced, BRCA1-dependent, clearance of DNA damage. Consequently, USP48 inactivation reduces chromosomal instability of FA-defective cells. Our results highlight a role for USP48 in controlling DNA repair and suggest it as a potential target that could be therapeutically exploited for FA.

The p14 ARF is a key tumor suppressor induced mainly by oncogenic stimuli. Although p14 ARF does not seem to respond to DNA damage, there are very few data regarding its role in other forms of stress, such as heat shock (HS) and oxidative stress (OS). Here, we report that suppression of p14 ARF increased resistance to cell death when cells were treated with H 2 O 2 or subjected to HS. In this setting, protection from cell death was mediated by elevated levels and activity of b-catenin, as downregulation of b-catenin alleviated the protective role of p14 ARF silencing. Moreover, Hsp70 was shown to regulate b-catenin protein levels by interacting with p14 ARF , suggesting that Hsp70, p14 ARF and b-catenin form a regulatory network. This novel pathway triggers cell death signals when cells are exposed to HS and OS.The p14 ARF tumor suppressor protein protects cells from malignant transformation by sensing various oncogenic signals 1,2 and activating p53-dependent checkpoint signals by interacting with MDM2, a p53 antagonist, within the nucleolus. 3,4 Although p14 ARF does not seem to respond to DNA damage, 5 there is limited information whether it could react to other forms of stress, such as heat shock (HS) and oxidative stress (OS). 6 Hsp70 is the major member of the 70-kDa HS protein family and its expression is dramatically increased after exposure to HS and OS. Induction of Hsp70 is accompanied by its translocation to the cell nucleus and particular to the nucleoli. 7,8 As a molecular chaperone, it is implicated in a number of cellular functions such as acquisition of thermoresistance, facilitation of protein translocation, regulation of protein degradation, antiapoptotic function and the protection of DNA from single strand breaks. [7][8][9][10][11][12] In addition, it is postulated that it behaves as a survival factor for cancer cells. 13,14 As p14 ARF and Hsp70 seem to function within the same subcellular organelle, the nucleoli, we set to examine whether p14 ARF plays a role in the regulation of the cellular response triggered by HS and OS. Material and Methods Cell lines and treatmentsHeLa, H1299 and NARF2 (a kind gift by Dr. Gordon Peters) cell lines were grown in Dulbeco's modified Eagle's medium supplemented with 10% fetal bovine serum and incubated at 37 C with 5% CO 2 . The p14 ARF -inducible NARF2 cell line is derived from U2OS cells and the induction of p14 ARF was achieved by the addition of 0.1 mM IPTG in the medium. HS was performed by incubation of the cells for 90 min either at 45 C (lethal temperature) for flow cytometric analysis or at 43.5 C for any other analysis. Cells were then left to recover for 24 hr. 15 For OS, cells were treated with 1 mM H 2 O 2 and then were left to recuperate for 24 hr. Plasmids and siRNA transfectionsThe phoenix amphotropic helper-free retrovirus producer line was used to construct retroviruses containing the pRE-TROSUPER-shp14 ARF Ref. 16 and the corresponding pRETROSUPERshLacz control. Infection of the HeLa and H1299 cells was performed at 12-hr intervals in 6...

Maintenance of genome integrity via repair of DNA damage is a key biological process required to suppress diseases, including Fanconi anemia (FA). We generated loss-of-function human haploid cells for FA complementation group C (FANCC), a gene encoding a component of the FA core complex, and used genome-wide CRISPR libraries as well as insertional mutagenesis to identify synthetic viable (genetic suppressor) interactions for FA. Here we show that loss of the BLM helicase complex suppresses FANCC phenotypes and we confirm this interaction in cells deficient for FA complementation group I and D2 (FANCI and FANCD2) that function as part of the FA I-D2 complex, indicating that this interaction is not limited to the FA core complex, hence demonstrating that systematic genome-wide screening approaches can be used to reveal genetic viable interactions for DNA repair defects.