Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.     

Alkylation DNA damage in combination with PARP inhibition results in formation of S-phase-dependent double-strand breaks

The combination of poly(ADP-ribose)polymerase (PARP) inhibitors and alkylating agents is currently being investigated in cancer therapy clinical trials. However, the DNA lesions producing the synergistic cell killing effect in tumors are not fully understood. Treatment of human and mouse fibroblasts with the monofunctional DNA methylating agent methyl methanesulfonate (MMS) in the presence of a PARP inhibitor has been shown to trigger a cell cycle checkpoint response. Among other changes, this DNA damage response to combination treatment includes activation of ATM/Chk2 and phosphorylation of histone H2A.X. These changes are consistent with DNA double-strand break (DSB) formation during the response, but the measurement of DSBs has not been addressed. Such DSB evaluation is important in understanding this DNA damage response because events other than DSB formation are known to lead to ATM/Chk2 activation and H2A.X phosphorylation. Here, we examined the structural integrity of genomic DNA after the combined treatment of cells with MMS and a PARP inhibitor, i.e., exposure to a sub-lethal dose of MMS in the presence of the PARP inhibitor 4-amino-1,8-napthalimide (4-AN). We used pulsed field gel electrophoresis (PFGE) for measurement of DSBs in both human and mouse embryonic fibroblasts, and flow cytometry to follow the phosphorylated form of H2A.X (γ-H2A.X). The results indicate that DSBs are formed with the combination treatment, but not following treatment with either agent alone. Our data also show that formation of γ-H2A.X correlates with PARP-1-expressing cells in S-phase of the cell cycle. The observations support the model that persistence of PARP-1 at base excision repair intermediates, as cells move into S-phase, leads to DSBs and the attendant checkpoint responses.     

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation,and impacts on metastasis free survival in breast cancer patients

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.     

A Stronger DNA Damage-Induced G2 Checkpoint Due to Over-Activated CHK1 in the Absence of PARP-1

Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in multi-pathways to respond to DNA damage. Lack of or inhibition of PARP-1 activity leads to slow progress of cell cycle and sensitization of cells to different stresses. Recently, it was reported that besides the Ku- dependent main non-homologous end joining (NHEJ) pathway, there is a PARP-1-dependent complementary NHEJ pathway to repair DNA double strand break (DSB). Here we show that compared with PARP-1+/+ cells, PARP-1-/- cells display a much stronger G2 checkpoint response following ionizing radiation (IR). Treatment with Chk1 siRNA abolishes the stronger G2 checkpoint response and sensitizes PARP-1-/- cells to IR. These data indicate that the stronger G2 checkpoint response in PARP-1-/- cells is CHK1-dependent, which protects cells from IR-induced killing. We also show that 4-Amino-1,8-naphthalimide (4-AN, inhibitor of PARP) but not methoxyamine (inhibitor of base excision repair (BER)), affects IR-induced G2 arrest and cell sensitivity in PARP-1+/+ cells, resulting in the phenotypes similar to those of PARP-1-/- cells. These results indicate that DSB repair from the complementary NHEJ pathway of PARP-1, but not single strand break (SSB) repair from the BER function of PARP-1, may play an essential role in the over-activated CHK1 regulated G2 checkpoint response and radiosensitivity in PARP-1-/- cells.     

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.     

The Saccharomyces cerevisiae RAD9, RAD17 and RAD24 genes are required for suppression of mutagenic post-replicative repair during chronic DNA damage

In Saccharomyces cerevisiae, a DNA damage checkpoint in the S-phase is responsible for delaying DNA replication in response to genotoxic stress. This pathway is partially regulated by the checkpoint proteins Rad9, Rad17 and Rad24. Here, we describe a novel hypermutable phenotype for rad9Δ, rad17Δ and rad24Δ cells in response to a chronic 0.01% dose of the DNA alkylating agent MMS. We report that this hypermutability results from DNA damage introduction during the S-phase and is dependent on a functional translesion synthesis pathway. In addition, we performed a genetic screen for interactions with rad9Δ that confer sensitivity to 0.01% MMS. We report and quantify 25 genetic interactions with rad9Δ, many of which involve the post-replication repair machinery. From these data, we conclude that defects in S-phase checkpoint regulation lead to increased reliance on mutagenic translesion synthesis, and we describe a novel role for members of the S-phase DNA damage checkpoint in suppressing mutagenic post-replicative repair in response to sublethal MMS treatment.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

Requirement for NBS1 in the S phase checkpoint response to DNA methylation combined with PARP inhibition

Treatment of PARP-1-expressing cells with the combination of a DNA methylating agent (MMS) and the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) leads to an ATR/Chk1-dependent S phase checkpoint and cell death by apoptosis. Activation of ATM/Chk2 is involved in sustaining the S phase checkpoint, and double strand break (DSB) accumulation was demonstrated. NBS1, part of the MRN complex that responds to DSBs, is known to modulate ATR- and ATM-dependent checkpoint responses to UV and IR, but a role in the response to PARP inhibition has not been addressed. Here we show that the S phase checkpoint observed 4-8h after MMS+4-AN treatment was absent in cells deficient in NBS1, but was present in NBS1-complemented (i.e., functionally wild-type) cells, indicating a critical role for NBS1 in this checkpoint response. NBS1 was phosphorylated in response to MMS+4-AN treatment, and this was partially ATR- and ATM-dependent, suggesting involvement of both upstream kinases. NBS1 expression had little effect on ATR-mediated phosphorylation of Chk1 and ATM-mediated phosphorylation of Chk2 in response to MMS+4-AN. Phosphorylation of SMC1 was also observed in response to MMS+4-AN treatment. In the absence of ATM and NBS1, phosphorylation of SMC1 was weak, especially at early times after MMS+4-AN treatment. In the absence of ATR activation, reduced SMC1 phosphorylation was seen over a 24h time course. These results suggested that both ATR and ATM phosphorylate SMC1 in response to MMS+4-AN and that this phosphorylation is enhanced by phospho-NBS1. The loss of the MMS+4-AN-induced S phase checkpoint in NBS1-deficient cells may be due to a reduced cellular level of the critical downstream effector, phospho-SMC1.     

PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA

Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1^−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1^−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.     

ATR signaling mediates an S-phase checkpoint after inhibition of poly(ADP-ribose) polymerase activity

Human fibroblasts, capable of expressing a kinase-dead form of ATR (ATRkd), can be sensitized to the cytotoxic effects of methyl methanesulfonate (MMS) by the PARP inhibitor 4-amino-1,8-naphthalimide (4-AN). The combination of MMS+4-AN results in accumulation of cells in S-phase of the cell cycle and activation of Chk1. Inhibition of ATR activity by expression of ATRkd suppresses the S-phase accumulation and partially reverses the Chk1 phosphorylation. The results confirm involvement of an ATR-mediated damage response pathway in the MMS+4-AN-induced S-phase cell cycle checkpoint in human fibroblasts. Consistent with this hypothesis, the inhibitors caffeine and UCN-01 also abrogate the ATR- and Chk1-mediated delay in progression through S-phase. In the absence of ATR-mediated signaling, MMS+4-AN exposure results in a G(2)/M arrest, rather than an S-phase checkpoint. Thus, whereas ATR mediates the S-phase response, it is not critical for arrest of cells in G(2)/M.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.     

The methyl methanesulfonate induced S-phase delay in XRCC1-deficient cells requires ATM and ATR

X-ray repair cross-complementing 1 (XRCC1) is required for DNA single-strand break and base excision repair (BER) in human cells. XRCC1-deficient human cells show hypersensitivity to cell killing, increased genetic instability and a significant delay in S-phase progression after exposure to the alkylating agent methyl methanesulfonate (MMS). Using RNAi modulation of XRCC1 levels, we show here that this S-phase delay is associated with significantly increased levels of recombinational repair as visualized by Rad51 focus formation. Using ATM- and ATR-defective cells and an ATM specific kinase inhibitor we demonstrate for the first time that the MMS-induced S-phase checkpoint requires both ATM and ATR. This unique dependency is associated with phosphorylation of ATM/ATR downstream targets or effectors such as SMC1 and Chk1. These results support the hypothesis that after MMS-treatment, the presence of unresolved BER intermediates gives rise to lesions that activate both ATM and ATR and that during the consequent S-phase delay, such intermediates may be repaired by a recombinational pathway which involves the Rad51 protein.     

The Pds1 anaphase inhibitor and Mec1 kinase define distinct checkpoints coupling S phase with mitosis in budding yeast

In most eukaryotic cells, DNA replication is confined to S phase of the cell cycle [1]. During this interval, S-phase checkpoint controls restrain mitosis until replication is complete [2]. In budding yeast, the anaphase inhibitor Pds1p has been associated with the checkpoint arrest of mitosis when DNA is damaged or when mitotic spindles have formed aberrantly [3] [4], but not when DNA replication is blocked with hydroxyurea (HU). Previous studies have implicated the protein kinase Mec1p in S-phase checkpoint control [5]. Unlike mec1 mutants, pds1 mutants efficiently inhibit anaphase when replication is blocked. This does not, however, exclude an essential S-phase checkpoint function of Pds1 beyond the early S-phase arrest point of a HU block. Here, we show that Pds1p is an essential component of a previously unsuspected checkpoint control system that couples the completion of S phase with mitosis. Further, the S-phase checkpoint comprises at least two distinct pathways. A Mec1p-dependent pathway operates early in S phase, but a Pds1p-dependent pathway becomes essential part way through S phase.     

Inhibition of poly (ADP-ribose) polymerase activates ATM which is required for subsequent homologous recombination repair

Poly (ADP-ribose) polymerase (PARP-1), ATM and DNA-dependent protein kinase (DNA-PK) are all involved in responding to DNA damage to activate pathways responsible for cellular survival. Here, we demonstrate that PARP-1^−/− cells are sensitive to the ATM inhibitor KU55933 and conversely that AT cells are sensitive to the PARP inhibitor 4-amino-1,8-napthalamide. In addition, PARP-1^−/− cells are shown to be sensitive to the DNA-PK inhibitor NU7026 and DNA-PKcs or Ku80 defective cells shown to be sensitive to PARP inhibitors. We believe PARP inhibition results in an increase in unresolved spontaneous DNA single-strand breaks (SSBs), which collapse replication forks and trigger homologous recombination repair (HRR). We show that ATM is activated following inhibition of PARP. Furthermore, PARP inhibitor-induced HRR is abolished in ATM, but not DNA-PK, inhibited cells. ATM and DNA-PK inhibition together give the same sensitivity to PARP inhibitors as ATM alone, indicating that ATM functions in the same pathways as DNA-PK for survival at collapsed forks, likely in non-homologous end joining (NHEJ). Altogether, we suggest that ATM is activated by PARP inhibitor-induced collapsed replication forks and may function upstream of HRR in the repair of certain types of double-strand breaks (DSBs).     

Replication-dependent cytotoxicity and Spartan-mediated repair of trapped PARP1–DNA complexes

The antitumor activity of poly(ADP-ribose) polymerase inhibitors (PARPis) has been ascribed to PARP trapping, which consists in tight DNA–protein complexes. Here we demonstrate that the cytotoxicity of talazoparib and olaparib results from DNA replication. To elucidate the repair of PARP1–DNA complexes associated with replication in human TK6 and chicken DT40 lymphoblastoid cells, we explored the role of Spartan (SPRTN), a metalloprotease associated with DNA replication, which removes proteins forming DPCs. We find that SPRTN-deficient cells are hypersensitive to talazoparib and olaparib, but not to veliparib, a weak PARP trapper. SPRTN-deficient cells exhibit delayed clearance of trapped PARP1 and increased replication fork stalling upon talazoparib and olaparib treatment. We also show that SPRTN interacts with PARP1 and forms nuclear foci that colocalize with the replicative cell division cycle 45 protein (CDC45) in response to talazoparib. Additionally, SPRTN is deubiquitinated and epistatic with translesion synthesis (TLS) in response to talazoparib. Our results demonstrate that SPRTN is recruited to trapped PARP1 in S-phase to assist in the excision and replication bypass of PARP1–DNA complexes.     

聚腺苷二磷酸-核糖聚合酶1功能与其抑制剂的作用及耐药机制

聚腺苷二磷酸-核糖聚合酶1(poly ADP-ribose polymerase-1,PARP1)是细胞中重要的修饰酶,其最广为人知的作用是通过自身PAR修饰,募集以XRCC1为首的多种DNA损伤修复效应蛋白质,参与DNA单、双链损伤修复。PARP1还能通过促进复制叉停滞与核小体解聚,为DNA损伤修复提供有利条件,维持基因组稳定性。近年来,除DNA损伤修复方面的作用,还发现PARP1能影响细胞凋亡、自噬与炎症通路,与神经退行性疾病的发生发展密切相关。而PARP抑制剂(PARP inhibitor,PARPi)是一种靶向PARP1,与细胞同源重组(homologous recombination,HR)缺陷表型共同作用,产生合成致死效应的抗肿瘤药物。该药物可捕获PARP1并抑制其活性,一方面直接干扰PARP1参与的DNA损伤修复通路,另一方面也抑制了PARP1介导的DNA损伤修复通路选择和复制叉停滞,使细胞基因组不稳定。然而,在临床治疗中常发现肿瘤细胞对PARPi不敏感。肿瘤细胞对PARPi耐药与自身基因突变高度相关,这些基因分别作用于细胞HR修复途径、PARP1循环途径、复制叉稳定性和药物主动外排等方面,在耐药肿瘤患者中确定具体的突变位点,将为临床治疗提供帮助。本文旨在对PARP1的功能作一综述,并重点介绍PARPi的作用机制和与肿瘤耐药相关的突变基因及其耐药机制,以期加深对细胞中PARP1介导的DNA损伤修复通路的认识,并为将来的临床治疗提供新思路。     

Drug-sensitive DNA polymerase δ reveals a role for mismatch repair in checkpoint activation in yeast

We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.     

Interaction between PARP-1 and ATR in mouse fibroblasts is blocked by PARP inhibition

Inhibition of PARP activity results in extreme sensitization to MMS-induced cell killing in cultured mouse fibroblasts. In these MMS-treated cells, PARP inhibition is accompanied by an accumulation of S-phase cells that requires signaling by the checkpoint kinase ATR [J.K. Horton, D.F. Stefanick, J.M. Naron, P.S. Kedar, S.H. Wilson, Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest following DNA methylating agent exposure, J. Biol. Chem. 280 (2005) 15773-15785]. Here, we examined mouse fibroblast extracts for formation of a complex that may reflect association between the damage responsive proteins PARP-1 and ATR. Co-immunoprecipitation of PARP-1 and ATR was observed in extracts prepared from MMS-treated cells, but not under conditions of PARP inhibition. Further, our experiments demonstrated PAR-adduction of ATR in extracts from control and MMS-treated cells. An interaction between purified ATR and PARP-1 was similarly demonstrated, suggesting that the observed co-immunoprecipitation of ATR and PARP-1 from cell extracts may be due to a direct interaction between the two enzymes. In addition, purified recombinant ATR is a substrate for poly(ADP-ribosyl)ation by PARP-1, and poly(ADP-ribose) adduction of PARP-1 and ATR resulted in an increase in PARP-1 and ATR co-immunoprecipitation.