A robust dual gene ON-OFF toggle directed by two independent promoter-degron pairs.

Switching genes on and off on cue is a cornerstone for understanding gene functions. One contemporary approach for loss-of-function studies of essential genes involves CRISPR-mediated knockout of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect in mammalian cell lines. A broadening of this approach would involve simultaneously switching on a second construct to interrogate the functions of a gene in the pathway. In this study, we developed a pair of switches that were independently controlled by both inducible promoters and degrons, enabling the toggling between two constructs with comparable kinetics and tightness. The gene-OFF switch was based on TRE transcriptional control coupled with auxin-induced degron-mediated proteolysis. A second independently controlled gene-ON switch was based on a modified ecdysone promoter and mutated FKBP12-derived destabilization domain degron, allowing acute and tuneable gene activation. This platform facilitates efficient generation of knockout cell lines containing a two-gene switch that is regulated tightly and can be flipped within a fraction of the time of a cell cycle.

Cyclin A-CDK1 suppresses the expression of the CDK1 activator CDC25A to safeguard timely mitotic entry.

Cyclin A and CDC25A are both activators of cyclin-dependent kinases (CDKs): cyclin A acts as an activating subunit of CDKs and CDC25A a phosphatase of the inhibitory phosphorylation sites of the CDKs. In this study, we uncovered an inverse relationship between the two CDK activators. As cyclin A is an essential gene, we generated a conditional silencing cell line using a combination of CRISPR-Cas9 and degron-tagged cyclin A. Destruction of cyclin A promoted an acute accumulation of CDC25A. The increase of CDC25A after cyclin A depletion occurred throughout the cell cycle and was independent on cell cycle delay caused by cyclin A deficiency. Moreover, we determined that the inverse relationship with cyclin A was specific for CDC25A and not for other CDC25 family members or kinases that regulate the same sites in CDKs. Unexpectedly, the upregulation of CDC25A was mainly caused by an increase in transcriptional activity instead of a change in the stability of the protein. Reversing the accumulation of CDC25A severely delayed G2-M in cyclin A-depleted cells. Taken together, these data provide evidence of a compensatory mechanism involving CDC25A that ensures timely mitotic entry at different levels of cyclin A.

Timeless Interacts with PARP-1 to Promote Homologous Recombination Repair.

Human Timeless helps stabilize replication forks during normal DNA replication and plays a critical role in activation of the S phase checkpoint and proper establishment of sister chromatid cohesion. However, it remains elusive whether Timeless is involved in the repair of damaged DNA. Here, we identify that Timeless physically interacts with PARP-1 independent of poly(ADP-ribosyl)ation. We present high-resolution crystal structures of Timeless PAB (PARP-1-binding domain) in free form and in complex with PARP-1 catalytic domain. Interestingly, Timeless PAB domain specifically recognizes PARP-1, but not PARP-2 or PARP-3. Timeless-PARP-1 interaction does not interfere with PARP-1 enzymatic activity. We demonstrate that rapid and transient accumulation of Timeless at laser-induced DNA damage sites requires PARP-1, but not poly(ADP-ribosyl)ation and that Timeless is co-trapped with PARP-1 at DNA lesions upon PARP inhibition. Furthermore, we show that Timeless and PARP-1 interaction is required for efficient homologous recombination repair.

Whole-Genome Duplication and Genome Instability in Cancer Cells: Double the Trouble.

Whole-genome duplication (WGD) is one of the most common genomic abnormalities in cancers. WGD can provide a source of redundant genes to buffer the deleterious effect of somatic alterations and facilitate clonal evolution in cancer cells. The extra DNA and centrosome burden after WGD is associated with an elevation of genome instability. Causes of genome instability are multifaceted and occur throughout the cell cycle. Among these are DNA damage caused by the abortive mitosis that initially triggers tetraploidization, replication stress and DNA damage associated with an enlarged genome, and chromosomal instability during the subsequent mitosis in the presence of extra centrosomes and altered spindle morphology. Here, we chronicle the events after WGD, from tetraploidization instigated by abortive mitosis including mitotic slippage and cytokinesis failure to the replication of the tetraploid genome, and finally, to the mitosis in the presence of supernumerary centrosomes. A recurring theme is the ability of some cancer cells to overcome the obstacles in place for preventing WGD. The underlying mechanisms range from the attenuation of the p53-dependent G1 checkpoint to enabling pseudobipolar spindle formation via the clustering of supernumerary centrosomes. These survival tactics and the resulting genome instability confer a subset of polyploid cancer cells proliferative advantage over their diploid counterparts and the development of therapeutic resistance.

Dovitinib induces mitotic defects and activates the G2 DNA damage checkpoint.

Dovitinib (TKI258; formerly CHIR-258) is an orally bioavailable inhibitor of multiple receptor tyrosine kinases. Interestingly, Dovitinib triggered a G2 /M arrest in cancer cell lines from diverse origins including HeLa, nasopharyngeal carcinoma, and hepatocellular carcinoma. Single-cell analysis revealed that Dovitinib promoted a delay in mitotic exit in a subset of cells, causing the cells to undergo mitotic slippage. Higher concentrations of Dovitinib induced a G2 arrest similar to the G2 DNA damage checkpoint. In support of this, DNA damage was triggered by Dovitinib as revealed by γ-H2AX and comet assays. The mitotic kinase CDK1 was found to be inactivated by phosphorylation in the presence of Dovitinib. Furthermore, the G2 arrest could be overcome by abrogation of the G2 DNA damage checkpoint using small molecule inhibitors of CHK1 and WEE1. Finally, Dovitinib-mediated G2 cell cycle arrest and subsequent cell death could be promoted after DNA damage repair was disrupted by inhibitors of poly(ADP-ribose) polymerases. These results are consistent with the recent finding that Dovitinib can also target topoisomerases. Collectively, these results suggest additional directions for use of Dovitinib, in particular with agents that target the DNA damage checkpoint.

G1 checkpoint establishment in vivo during embryonic liver development.

BACKGROUND: The DNA damage-mediated cell cycle checkpoint is an essential mechanism in the DNA damage response (DDR). During embryonic development, the characteristics of cell cycle and DNA damage checkpoint evolve from an extremely short G1 cell phase and lacking G1 checkpoint to lengthening G1 phase and the establishment of the G1 checkpoint. However, the regulatory mechanisms governing these transitions are not well understood. In this study, pregnant mice were exposed to ionizing radiation (IR) to induce DNA damage at different embryonic stages; the kinetics and mechanisms of the establishment of DNA damage-mediated G1 checkpoint in embryonic liver were investigated. RESULTS: We found that the G2 cell cycle arrest was the first response to DNA damage in early developmental stages. Starting at E13.5/E15.5, IR mediated inhibition of the G1 to S phase transition became evident. Concomitantly, IR induced the robust expression of p21 and suppressed Cdk2/cyclin E activity, which might involve in the initiation of G1 checkpoint. The established G1 cell cycle checkpoint, in combination with an enhanced DNA repair capacity at E15.5, displayed biologically protective effects of repairing DNA double-strand breaks (DSBs) and reducing apoptosis in the short term as well as reducing chromosome deletion and breakage in the long term. CONCLUSION: Our study is the first to demonstrate the establishment of the DNA damage-mediated G1 cell cycle checkpoint in liver cells during embryogenesis and its in vivo biological effects during embryonic liver development.

Coregulation of NDC80 Complex Subunits Determines the Fidelity of the Spindle-Assembly Checkpoint and Mitosis.

NDC80 complex (NDC80C) is composed of four subunits (SPC24, SPC25, NDC80, and NUF2) and is vital for kinetochore-microtubule (KT-MT) attachment during mitosis. Paradoxically, NDC80C also functions in the activation of the spindle-assembly checkpoint (SAC). This raises an interesting question regarding how mitosis is regulated when NDC80C levels are compromised. Using a degron-mediated depletion system, we found that acute silencing of SPC24 triggered a transient mitotic arrest followed by mitotic slippage. SPC24-deficient cells were unable to sustain SAC activation despite the loss of KT-MT interaction. Intriguingly, our results revealed that other subunits of the NDC80C were co-downregulated with SPC24 at a posttranslational level. Silencing any individual subunit of NDC80C likewise reduced the expression of the entire complex. We found that the SPC24-SPC25 and NDC80-NUF2 subcomplexes could be individually stabilized using ectopically expressed subunits. The synergism of SPC24 downregulation with drugs that promote either mitotic arrest or mitotic slippage further underscored the dual roles of NDC80C in KT-MT interaction and SAC maintenance. The tight coordinated regulation of NDC80C subunits suggests that targeting individual subunits could disrupt mitotic progression and provide new avenues for therapeutic intervention. IMPLICATIONS: These results highlight the tight coordinated regulation of NDC80C subunits and their potential as targets for antimitotic therapies.

BCL-XL regulates the timing of mitotic apoptosis independently of BCL2 and MCL1 compensation.

Mitotic catastrophe induced by prolonged mitotic arrest is a major anticancer strategy. Although antiapoptotic BCL2-like proteins, including BCL-XL, are known to regulate apoptosis during mitotic arrest, adaptive changes in their expression can complicate loss-of-function studies. Our studies revealed compensatory alterations in the expression of BCL2 and MCL1 when BCL-XL is either downregulated or overexpressed. To circumvent their reciprocal regulation, we utilized a degron-mediated system to acutely silence BCL-XL just before mitosis. Our results show that in epithelial cell lines including HeLa and RPE1, BCL-XL and BCL2 acted collaboratively to suppress apoptosis during both unperturbed cell cycle and mitotic arrest. By tagging BCL-XL and BCL2 with a common epitope, we estimated that BCL-XL was less abundant than BCL2 in the cell. Nonetheless, BCL-XL played a more prominent antiapoptotic function than BCL2 during interphase and mitotic arrest. Loss of BCL-XL led to mitotic cell death primarily through a BAX-dependent process. Furthermore, silencing of BCL-XL led to the stabilization of MCL1, which played a significant role in buffering apoptosis during mitotic arrest. Nevertheless, even in a MCL1-deficient background, depletion of BCL-XL accelerated mitotic apoptosis. These findings underscore the pivotal involvement of BCL-XL in controlling timely apoptosis during mitotic arrest, despite adaptive changes in the expression of other BCL2-like proteins.

Depletion of p31comet protein promotes sensitivity to antimitotic drugs.

Antimitotic spindle poisons are among the most important chemotherapeutic agents available. However, precocious mitotic exit by mitotic slippage limits the cytotoxicity of spindle poisons. The MAD2-binding protein p31(comet) is implicated in silencing the spindle assembly checkpoint after all kinetochores are attached to spindles. In this study, we report that the levels of p31(comet) and MAD2 in different cell lines are closely linked with susceptibility to mitotic slippage. Down-regulation of p31(comet) increased the sensitivity of multiple cancer cell lines to spindle poisons, including nocodazole, vincristine, and Taxol. In the absence of p31(comet), lower concentrations of spindle poisons were required to induce mitotic block. The delay in checkpoint silencing was induced by an accumulation of mitotic checkpoint complexes. The increase in the duration of mitotic block after p31(comet) depletion resulted in a dramatic increase in mitotic cell death upon challenge with spindle poisons. Significantly, cells that are normally prone to mitotic slippage and resistant to spindle disruption-mediated mitotic death were also sensitized after p31(comet) depletion. These results highlight the importance of p31(comet) in checkpoint silencing and its potential as a target for antimitotic therapies.

SCFß(TrCP) mediates stress-activated MAPK-induced Cdc25B degradation.

Cdc25A, which is one of the three mammalian CDK-activating Cdc25 protein phosphatases (Cdc25A, B and C), is degraded through SCF(ßTrCP)-mediated ubiquitylation following genomic insult; however, the regulation of the stability of the other two Cdc25 proteins is not well understood. Previously, we showed that Cdc25B is primarily degraded by cellular stresses that activate stress-activated MAPKs, such as Jun NH(2)-terminal kinase (JNK) and p38. Here, we report that Cdc25B was ubiquitylated by SCF(ßTrCP) E3 ligase upon phosphorylation at two Ser residues in the ßTrCP-binding-motif-like sequence D(94)AGLCMDSPSP(104). Point mutation of these Ser residues to alanine (Ala) abolished the JNK-induced ubiquitylation by SCF(ßTrCP), and point mutation of DAG to AAG or DAA eradicated both ßTrCP binding and ubiquitylation. Further analysis of the mode of ßTrCP binding to this region revealed that the PEST-like sequence from E(82)SS to D(94)AG is crucially involved in both the ßTrCP binding and ubiquitylation of Cdc25B. Furthermore, the phospho-mimetic replacement of all 10 Ser residues in the E(82)SS to SPSP(104) region with Asp resulted in ßTrCP binding. Collectively, these results indicate that stress-induced Cdc25B ubiquitylation by SCF(ßTrCP) requires the phosphorylation of S(101)PS(103)P in the ßTrCP-binding-motif-like and adjacent PEST-like sequences.

MARCH5 regulates mitotic apoptosis through MCL1-dependent and independent mechanisms.

The anti-apoptotic MCL1 is critical for delaying apoptosis during mitotic arrest. MCL1 is degraded progressively during mitotic arrest, removing its anti-apoptotic function. We found that knockout of components of ubiquitin ligases including APC/C, SCF complexes, and the mitochondrial ubiquitin ligase MARCH5 did not prevent mitotic degradation of MCL1. Nevertheless, MARCH5 determined the initial level of MCL1-NOXA network upon mitotic entry and hence the window of time during MCL1 was present during mitotic arrest. Paradoxically, although knockout of MARCH5 elevated mitotic MCL1, mitotic apoptosis was in fact enhanced in a BAK-dependent manner. Mitotic apoptosis was accelerated after MARCH5 was ablated in both the presence and absence of MCL1. Cell death was not altered after disrupting other MARCH5-regulated BCL2 family members including NOXA, BIM, and BID. Disruption of the mitochondrial fission factor DRP1, however, reduced mitotic apoptosis in MARCH5-disrupted cells. These data suggest that MARCH5 regulates mitotic apoptosis through MCL1-independent mechanisms including mitochondrial maintenance that can overcome the stabilization of MCL1.

Orderly inactivation of the key checkpoint protein mitotic arrest deficient 2 (MAD2) during mitotic progression.

Anaphase is promoted by the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) only when all the chromosomes have achieved bipolar attachment to the mitotic spindles. Unattached kinetochores or the absence of tension between the paired kinetochores activates a surveillance mechanism termed the spindle-assembly checkpoint. A fundamental principle of the checkpoint is the activation of mitotic arrest deficient 2 (MAD2). MAD2 then forms a diffusible complex called mitotic checkpoint complex (designated as MAD2(MCC)) before it is recruited to APC/C (designated as MAD2(APC/C)). Large gaps in our knowledge remain on how MAD2 is inactivated after the checkpoint is satisfied. In this study, we have investigated the regulation of MAD2-containing complexes during mitotic progression. Using selective immunoprecipitation of checkpoint components and gel filtration chromatography, we found that MAD2(MCC) and MAD2(APC/C) were regulated very differently during mitotic exit. Temporally, MAD2(MCC) was broken down ahead of MAD2(APC/C). The inactivation of the two complexes also displayed different requirements of proteolysis; although APC/C and proteasome activities were dispensable for MAD2(MCC) inactivation, they are required for MAD2(APC/C) inactivation. In fact, the degradation of CDC20 is inextricably linked to the breakdown of MAD2(APC/C). These data extended our understanding of the checkpoint complexes during checkpoint silencing.

How protein kinases co-ordinate mitosis in animal cells.

Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

Cell Cycle Control: A System of Interlinking Oscillators.

The cell cycle is the sequence of events through which a cell duplicates its genome, grows, and divides. Key cell cycle transitions are driven by oscillators comprising of protein kinases and their regulators. Different cell cycle oscillators are inextricably linked to ensure orderly activation of oscillators. A recurring theme in their regulation is the abundance of autoamplifying loops that ensure switch-like and unidirectional cell cycle transitions. The periodicity of many cell cycle oscillators is choreographed by inherent mechanisms that promote automatic inactivation, often involving dephosphorylation and ubiquitin-mediated protein degradation. These inhibitory signals are subsequently suppressed to enable the next cell cycle to occur. Although the activation and inactivation of cell cycle oscillators are in essence autonomous during the unperturbed cell cycle, a number of checkpoint mechanisms are able to halt the cell cycle until preconditions or defects are addressed. Together, these mechanisms orchestrate orderly progression of the cell cycle to produce more cells and to safeguard genome stability.

One-step multiplex toolkit for efficient generation of conditional gene silencing human cell lines.

Loss-of-function analysis is one of the major arsenals we have for understanding gene functions in mammalian cells. For analysis of essential genes, the major challenge is to develop simple methodologies for tight and rapid inducible gene inactivation. One approach involves CRISPR-Cas9-mediated disruption of the endogenous locus in conjunction with the expression of a rescue construct, which can subsequently be turned off to produce a gene inactivation effect. Here we describe the development of a set of Sleeping Beauty transposon-based vectors for expressing auxin-inducible degron (AID)-tagged genes under the regulation of a tetracycline-controlled promoter. The dual transcriptional and degron-mediated post-translational regulation allows rapid and tight silencing of protein expression in mammalian cells. We demonstrated that both non-essential and essential genes could be targeted in human cell lines using a one-step transfection method. Moreover, multiple genes could be simultaneously or sequentially targeted, allowing inducible inactivation of multiple genes. These resources enable highly efficient generation of conditional gene silencing cell lines to facilitate functional studies of essential genes.

DNA damage mediated s and g(2) checkpoints in human embryonal carcinoma cells.

For mouse embryonic stem (ES) cells, the importance of the S and G(2) cell cycle checkpoints for genomic integrity is increased by the absence of the G(1) checkpoint. We have investigated ionizing radiation (IR)-mediated cell cycle checkpoints in undifferentiated and retinoic acid-differentiated human embryonal carcinoma (EC) cells. Like mouse ES cells, human EC cells did not undergo G(1) arrest after IR but displayed a prominent S-phase delay followed by a G(2)-phase delay. In contrast, although differentiated EC cells also failed to arrest at G(1)-phase after IR, they quickly exited S-phase and arrested in G(2)-phase. In differentiated EC cells, the G(2)-M-phase cyclin B1/CDC2 complex was upregulated after IR, but the G(1)-S-phase cyclin E and the cyclin E/CDK2 complex were expressed at constitutively low levels, which could be an important factor distinguishing DNA damage responses between undifferentiated and differentiated EC cells. S-phase arrest and expression of p21 could be inhibited by 7-hydroxystaurosporine, suggesting that the ataxia-telangiectasia and Rad-3-related-checkpoint kinase 1 (ATR-CHK1), and p21 pathways might play a role in the IR-mediated S-phase checkpoint in EC cells. IR-mediated phosphorylation of ataxia-telangiectasia mutated, (CHK1), and checkpoint kinase 2 were distinctly higher in undifferentiated EC cells compared with differentiated EC cells. Combined with the prominent S and G(2) checkpoints and a more efficient DNA damage repair system, these mechanisms operate together in the maintenance of genome stability for EC cells.

DNA damage and polyploidization.

A growing body of evidence indicates that polyploidization triggers chromosomal instability and contributes to tumorigenesis. DNA damage is increasingly being recognized for its roles in promoting polyploidization. Although elegant mechanisms known as the DNA damage checkpoints are responsible for halting the cell cycle after DNA damage, agents that uncouple the checkpoints can induce unscheduled entry into mitosis. Likewise, defects of the checkpoints in several disorders permit mitotic entry even in the presence of DNA damage. Forcing cells with damaged DNA into mitosis causes severe chromosome segregation defects, including lagging chromosomes, chromosomal fragments and chromosomal bridges. The presence of these lesions in the cleavage plane is believed to abort cytokinesis. It is postulated that if cytokinesis failure is coupled with defects of the p53-dependent postmitotic checkpoint pathway, cells can enter S phase and become polyploids. Progress in the past several years has unraveled some of the underlying principles of these pathways and underscored the important role of DNA damage in polyploidization. Furthermore, polyploidization per se may also be an important determinant of sensitivity to DNA damage, thereby may offer an opportunity for novel therapies.

Specialized roles of the two mitotic cyclins in somatic cells: cyclin A as an activator of M phase-promoting factor.

The role of cyclin B-CDC2 as M phase-promoting factor (MPF) is well established, but the precise functions of cyclin A remain a crucial outstanding issue. Here we show that down-regulation of cyclin A induces a G2 phase arrest through a checkpoint-independent inactivation of cyclin B-CDC2 by inhibitory phosphorylation. The phenotype is rescued by expressing cyclin A resistant to the RNA interference. In contrast, down-regulation of cyclin B disrupts mitosis without inactivating cyclin A-CDK, indicating that cyclin A-CDK acts upstream of cyclin B-CDC2. Even when ectopically expressed, cyclin A cannot replace cyclin B in driving mitosis, indicating the specific role of cyclin B as a component of MPF. Deregulation of WEE1, but not the PLK1-CDC25 axis, can override the arrest caused by cyclin A knockdown, suggesting that cyclin A-CDK may tip the balance of the cyclin B-CDC2 bistable system by initiating the inactivation of WEE1. These observations show that cyclin A cannot form MPF independent of cyclin B and underscore a critical role of cyclin A as a trigger for MPF activation.

Aurora kinases and DNA damage response.

It is well established that Aurora kinases perform critical functions during mitosis. It has become increasingly clear that the Aurora kinases also perform a myriad of non-mitotic functions including DNA damage response. The available evidence indicates that inhibition Aurora kinase A (AURKA) may contribute to the G2 DNA damage checkpoint through AURKA's functions in PLK1 and CDC25B activation. Both AURKA and Aurora kinase B (AURKB) are also essential in mitotic DNA damage response that guard against DNA damage-induced chromosome segregation errors, including the control of abscission checkpoint and prevention of micronuclei formation. Dysregulation of Aurora kinases can trigger DNA damage in mitosis that is sensed in the subsequent G1 by a p53-dependent postmitotic checkpoint. Aurora kinases are themselves linked to the G1 DNA damage checkpoint through p53 and p73 pathways. Finally, several lines of evidence provide a connection between Aurora kinases and DNA repair and apoptotic pathways. Although more studies are required to provide a comprehensive picture of how cells respond to DNA damage, these findings indicate that both AURKA and AURKB are inextricably linked to pathways guarding against DNA damage. They also provide a rationale to support more detailed studies on the synergism between small-molecule inhibitors against Aurora kinases and DNA-damaging agents in cancer therapies.

Synergism between ATM and PARP1 Inhibition Involves DNA Damage and Abrogating the G2 DNA Damage Checkpoint.

PARP inhibitors have emerged as effective chemotherapeutic agents for BRCA1/BRCA2-deficient cancers. Another DNA damage response protein, ATM, is also increasingly being recognized as a target for synthetic lethality with PARP inhibitors. As ATM functions in both cell cycle arrest and DNA repair after DNA damage, how cells respond to inhibition of ATM and PARP1 is yet to be defined precisely. We found that loss of ATM function, either in an ATM-deficient background or after treatment with ATM inhibitors (KU-60019 or AZD0156), results in spontaneous DNA damage and an increase in PARylation. When PARP1 is also deleted or inhibited with inhibitors (olaparib or veliparib), the massive increase in DNA damage activates the G2 DNA damage checkpoint kinase cascade involving ATR, CHK1/2, and WEE1. Our data indicated that the role of ATM in DNA repair is critical for the synergism with PARP inhibitors. Bypass of the G2 DNA damage checkpoint in the absence of ATM functions occurs only after a delay. The relative insensitivity of PARP1-deficient cells to PARP inhibitors suggested that other PARP isoforms played a relatively minor role in comparison with PARP1 in synergism with ATMi. As deletion of PARP1 also increased sensitivity to ATM inhibitors, trapping of PARP1 on DNA may not be the only mechanism involved in the synergism between PARP1 and ATM inhibition. Collectively, these studies provide a mechanistic foundation for therapies targeting ATM and PARP1.