The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted

DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.     

The DNA crosslink-induced S-phase checkpoint depends on ATR-CHK1 and ATR-NBS1-FANCD2 pathways

The genetic syndrome Fanconi anemia (FA) is characterized by aplastic anemia, cancer predisposition and hypersensitivity to DNA interstrand crosslinks (ICLs). FA proteins (FANCs) are thought to work in pathway(s) essential for dealing with crosslinked DNA. FANCs interact with other proteins involved in both DNA repair and S-phase checkpoint such as BRCA1, ATM and the RAD50/MRE11/NBS1 (RMN) complex. We deciphered the previously undefined pathway(s) leading to the ICLs-induced S-phase checkpoint and the role of FANCs in this process. We found that ICLs activate a branched pathway downstream of the ATR kinase: one branch depending on CHK1 activity and the other on the FANCs-RMN complex. The transient slow-down of DNA synthesis was abolished in cells lacking ATR, whereas CHK1-siRNA-treated cells, NBS1 or FA cells showed partial S-phase arrest. CHK1 RNAi in NBS1 or FA cells abolished the S-phase checkpoint, suggesting that CHK1 and FANCs/NBS1 proteins work on parallel pathways. Furthermore, we found that ICLs trigger ATR-dependent FANCD2 phosphorylation and FANCD2/ATR colocalization. This study demonstrates a novel relationship between the FA pathway(s) and the ATR kinase.     

Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response

Pyrrole–imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition.     

A p53-dependent checkpoint pathway prevents rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.     

Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision

Correct regulation of the replication licensing system ensures that no DNA is rereplicated in a single cell cycle. When the licensing protein Cdt1 is overexpressed in G2 phase of the cell cycle, replication origins are relicensed and the DNA is rereplicated. At the same time, checkpoint pathways are activated that block further cell cycle progression. We have studied the consequence of deregulating the licensing system by adding recombinant Cdt1 to Xenopus egg extracts. We show that Cdt1 induces checkpoint activation and the appearance of small fragments of double-stranded DNA. DNA fragmentation and strong checkpoint activation are dependent on uncontrolled rereplication and do not occur after a single coordinated round of rereplication. The DNA fragments are composed exclusively of rereplicated DNA. The unusual characteristics of these fragments suggest that they result from head-to-tail collision (rear ending) of replication forks chasing one another along the same DNA template.     

Activation of Fanconi Anemia Pathway in Cells with Re-Replicated DNA

To maintain genome stability, the cell has to limit initiation of DNA replication to once per cell cycle. Loss of this control leads to DNA re-replication with repeated firing of replication origins in the same cell cycle. Re-replication generates both ssDNA and double strand breaks, as well as activation of the DNA damage checkpoint. In re-replicated cells, activation of the checkpoint is critical to arrest cells in G2 resulting in accumulation of cells with re-replicated DNA. Abrogation of this checkpoint suppresses the progressive accumulation of cells with excess DNA and causes apoptosis. Recently, the Fanconi Anemia pathway was reported to be activated in re-replicating cells. Interestingly, FA core complexes but not FANCD2, is required for checkpoint activation in re-replicated cells, suggesting that the pathway to checkpoint activation requires the ubiquitination of substrates other than FANCD2. In addition, FANCD2 is required for recruitment of Rad51 to foci in re-replicated cells, so that the repair pathways activated after small degrees of re-replication are expected to be compromised in cells with mutations in the FA pathway.     

Distinct roles for DNA-PK,ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress

DNA damage encountered by DNA replication forks poses risks of genome destabilization, a precursor to carcinogenesis. Damage checkpoint systems cause cell cycle arrest, promote repair and induce programed cell death when damage is severe. Checkpoints are critical parts of the DNA damage response network that act to suppress cancer. DNA damage and perturbation of replication machinery causes replication stress, characterized by accumulation of single-stranded DNA bound by replication protein A (RPA), which triggers activation of ataxia telangiectasia and Rad3 related (ATR) and phosphorylation of the RPA32, subunit of RPA, leading to Chk1 activation and arrest. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) [a kinase related to ataxia telangiectasia mutated (ATM) and ATR] has well characterized roles in DNA double-strand break repair, but poorly understood roles in replication stress-induced RPA phosphorylation. We show that DNA-PKcs mutant cells fail to arrest replication following stress, and mutations in RPA32 phosphorylation sites targeted by DNA-PKcs increase the proportion of cells in mitosis, impair ATR signaling to Chk1 and confer a G2/M arrest defect. Inhibition of ATR and DNA-PK (but not ATM), mimic the defects observed in cells expressing mutant RPA32. Cells expressing mutant RPA32 or DNA-PKcs show sustained H2AX phosphorylation in response to replication stress that persists in cells entering mitosis, indicating inappropriate mitotic entry with unrepaired damage.     

Complex role of the FA proteins in providing genome stability

FA is a rare genetic disorder characterized by developmental abnormalities, bone marrow failure and cancer susceptibility. Cells that are derived from patients with FA display spontaneous chromosomal instability and hypersensitivity to DNA crosslinking agents that is used in FA clinical diagnostics. FA is genetically heterogeneous and caused by mutations in at least 11 distinct genes, FANCA, FANCA, B, C, D1, D2, E, F, G, I, J and L. FA proteins interact with various proteins involved in DNA damage response and cell cycle checkpoint regulation, such as: RAD51, BRCA1, BRCA2, ATM or NBS1. Moreover, BRCA2 that plays a crucial role in homologous recombination is one of FA proteins. Collectively, all these data indicate, that the FA pathway is involved in different molecular processes that prevent DNA and control genomic stability, although its precise role still remains undefined.     

Human CDK2 Inhibition Modifies the Dynamics of Chromatin-Bound Minichromosome Maintenance Complex and Replication Protein A

Minichromosome maintenance (MCM) proteins form a complex and possess helicase activity to unwind the DNA duplex and establish a replication fork. To assure that origins only fire once per cell cycle, the MCM complex is removed from chromatin and inactivated as cells exit S phase. In this report, we demonstrate that CDK2 depletion in human cells leads to an overall phosphorylation defect at mitosis with increased rereplication, correlated with the accumulation of chromatin-bound MCM proteins. We show that CDK2 suppression results in decreased MCM4 phosphorylation at multiple serine and threonine sites. In addition, CDK2 inhibition induces an increase in chromatin-bound replication protein A (RPA) which should bindto single-stranded DNA regions, possibly establishing a replication intermediate that activates the ATR cascade. Finally, we observe that loss of CDK2 function in G1 delays replication initiation while it promotes rereplication in G2/M. Thus, by modulating the phospho-status of MCM4 and regulating origin firing, S phase CDK2 appears to be an integrated component of cellular machinery required for temporally controlling replication activity and maintaining genomic stability.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G1/S checkpoint, ATM, and p53 signaling pathways in p53-wildtype cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G1/S checkpoint pathway in p53-wildtype lines and not in p53-mutant cells. These responses are coupled with G2/G1 checkpoint effectors p21CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G2 cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wildtype and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G2 arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wildtype and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell-cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.     

ATR pathway is the primary pathway for activating G2/M checkpoint induction after re-replication

DNA replication is tightly controlled to ensure accurate chromosome duplication and segregation in each cell cycle. Inactivation of Geminin, an inhibitor of origin licensing, leads to re-replication in human tumor cells within the same cell cycle and triggers a G(2)/M checkpoint. We find that the primary pathway to signal that re-replication has been detected is the ATR kinase and the Rad9-Rad1-Hus1 (9-1-1) clamp complex together with Rad17-RFC clamp loader. ATM kinase and the Mre11-Rad50-Nbs1 complex do not appear to play significant roles in the checkpoint. Chk1 activation occurs at early stages, whereas Chk2 activation occurs much later. Overall we conclude that ATR/Chk1 pathway is activated at an early time point after the loss of Geminin and contributes to checkpoint arrest essential for the accumulation of re-replicated cells, whereas activation of the ATM/Chk2 pathway is a by-product of DNA re-replication at a later period.     

Loss of Geminin induces rereplication in the presence of functional p53

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.     

Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest evoked by pierisin-1

Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N(2) of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G(2)-M-phase delay. In the colony survival assays, Rad17(-/-) DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM(-/-) DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling.     

BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.     

Checkpoint defects leading to premature mitosis also cause endoreplication of DNA in Aspergillus nidulans

The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMX(cdc2) in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode a rad3/ATR homologue, whereas uvsD displays homology to rad26, which has only previously been identified in Schizosaccharomyces pombe. uvsB(rad3) and uvsD(rad26) have G2 checkpoint functions over mitosis and another function essential for surviving DNA damage. The rereplication phenotype is accompanied by lack of NIME(cyclinB), but ectopic expression of active nondegradable NIME(cyclinB) does not arrest DNA rereplication. DNA rereplication can also be induced in cells that enter mitosis prematurely because of lack of tyrosine phosphorylation of NIMX(cdc2) and impaired anaphase-promoting complex function. The data demonstrate that lack of checkpoint control over mitosis can secondarily cause defects in the checkpoint system that prevents DNA rereplication in the absence of mitosis. This defines a new mechanism by which endoreplication of DNA can be triggered and maintained in eukaryotic cells.     

FANCM and FAAP24 function in ATR-mediated checkpoint signaling independently of the Fanconi anemia core complex

The Fanconi anemia (FA) pathway is implicated in DNA repair and cancer predisposition. Central to this pathway is the FA core complex, which is targeted to chromatin by FANCM and FAAP24 following replication stress. Here we show that FANCM and FAAP24 interact with the checkpoint protein HCLK2 independently of the FA core complex. In addition to defects in FA pathway activation, downregulation of FANCM or FAAP24 also compromises ATR/Chk1-mediated checkpoint signaling, leading to defective Chk1, p53, and FANCE phosphorylation; 53BP1 focus formation; and Cdc25A degradation. As a result, FANCM and FAAP24 deficiency results in increased endogenous DNA damage and a failure to efficiently invoke cell-cycle checkpoint responses. Moreover, we find that the DNA translocase activity of FANCM, which is dispensable for FA pathway activation, is required for its role in ATR/Chk1 signaling. Our data suggest that DNA damage recognition and remodeling activities of FANCM and FAAP24 cooperate with ATR/Chk1 to promote efficient activation of DNA damage checkpoints.     

NER initiation factors,DDB2 and XPC,regulate UV radiation response by recruiting ATR and ATM kinases to DNA damage sites

ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.     

Crosstalk between BRCA‐Fanconi anemia and mismatch repair pathways prevents MSH2‐dependent aberrant DNA damage responses

Several proteins in the BRCA‐Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA‐FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA‐FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18‐dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2‐null primary mouse cells. Thus, we propose that regulation of MSH2‐dependent DNA damage response underlies the importance of interactions between BRCA‐FA and MMR pathways.