Cancer cells use self-inflicted DNA breaks to evade growth limits imposed by genotoxic stress.

Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.

The caspase-activated DNase: apoptosis and beyond.

Organismal development and function requires multiple and accurate signal transduction pathways to ensure that proper balance between cell proliferation, differentiation, inactivation, and death is achieved. Cell death via apoptotic caspase signal transduction is extensively characterized and integral to this balance. Importantly, the view of apoptotic signal transduction has expanded over the previous decades. Subapoptotic caspase signaling has surfaced as mechanism that can promote the adoption of a range of cellular fates. An emerging mechanism of subapoptotic caspase signaling is the activation of the caspase-activated DNase (CAD) through controlled cleavage of the inhibitor of CAD (ICAD). CAD-induced DNA breaks incite a DNA damage response, frequently invoking p53 signaling, that transduces a change in cell fate. Cell differentiation and senescence are fates demonstrated to arise from CAD-induced DNA breaks. Furthermore, an apparent consequence of CAD activity is also emerging, as a potential source of oncogenic mutations. This review will discuss the mechanisms underlying CAD-induced DNA breaks and highlight how CAD activity promotes diverse cell fates.

Temporal activation of XRCC1-mediated DNA repair is essential for muscle differentiation.

Transient DNA strand break formation has been identified as an effective means to enhance gene expression in living cells. In the muscle lineage, cell differentiation is contingent upon the induction of caspase-mediated DNA strand breaks, which act to establish the terminal gene expression program. This coordinated DNA nicking is rapidly resolved, suggesting that myoblasts may deploy DNA repair machinery to stabilize the genome and entrench the differentiated phenotype. Here, we identify the base excision repair pathway component XRCC1 as an indispensable mediator of muscle differentiation. Caspase-triggered XRCC1 repair foci form rapidly within differentiating myonuclei, and then dissipate as the maturation program proceeds. Skeletal myoblast deletion of Xrcc1 does not have an impact on cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. Together, these results demonstrate that XRCC1 manages a temporally responsive DNA repair process to advance the muscle differentiation program.

ATM/ATR-mediated phosphorylation of PALB2 promotes RAD51 function.

DNA damage activates the ATM and ATR kinases that coordinate checkpoint and DNA repair pathways. An essential step in homology-directed repair (HDR) of DNA breaks is the formation of RAD51 nucleofilaments mediated by PALB2-BRCA2; however, roles of ATM and ATR in this critical step of HDR are poorly understood. Here, we show that PALB2 is markedly phosphorylated in response to genotoxic stresses such as ionizing radiation and hydroxyurea. This response is mediated by the ATM and ATR kinases through three N-terminal S/Q-sites in PALB2, the consensus target sites for ATM and ATR Importantly, a phospho-deficient PALB2 mutant is unable to support proper RAD51 foci formation, a key PALB2 regulated repair event, whereas a phospho-mimicking PALB2 version supports RAD51 foci formation. Moreover, phospho-deficient PALB2 is less potent in HDR than wild-type PALB2. Further, this mutation reveals a separation in PALB2 function, as the PALB2-dependent checkpoint response is normal in cells expressing the phospho-deficient PALB2 mutant. Collectively, our findings highlight a critical importance of PALB2 phosphorylation as a novel regulatory step in genome maintenance after genotoxic stress.

Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control.

Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

CtIP-dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation.

To prevent accumulation of mutations, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homology-directed repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of ATR (ataxia telangiectasia mutated and Rad3 related) and CHK1 kinases to induce the cell cycle checkpoint. In this paper, we show that CHK1 was rapidly and robustly activated before detectable end resection. Moreover, we show that the key resection factor CtIP was dispensable for initial ATR-CHK1 activation after DNA damage by camptothecin and ionizing radiation. In contrast, we find that DNA end resection was critically required for sustained ATR-CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Consequently, resection-deficient cells entered mitosis with persistent DNA damage. In conclusion, we have uncovered a temporal program of checkpoint activation, where CtIP-dependent DNA end resection is required for sustained checkpoint signaling.

Parole terms for a killer: directing caspase3/CAD induced DNA strand breaks to coordinate changes in gene expression.

In a series of discoveries over the preceding decade, a number of laboratories have unequivocally established that apoptotic proteins and pathways are well conserved cell fate determinants, which act independent of a cell death response. Within this context, the role for apoptotic proteins in the induction of cell differentiation has been widely documented. Despite these discoveries, little information has been forthcoming regarding a conserved mechanism by which apoptotic proteins achieve this non-death outcome. In the following discussion, we will explore the premise that the penultimate step in apoptosis, genome wide DNA damage/strand breaks act as a conserved genomic reprogramming event necessary for cell differentiation (Larsen et al. Proc Natl Acad Sci USA 2010; 107:4230-5). Moreover, we hypothesis that directed DNA damage, as mediated by known apoptotic proteins, may participate in numerous forms of regulated gene expression.

Caspase 3/caspase-activated DNase promote cell differentiation by inducing DNA strand breaks.

Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes.