The distinctive cellular responses to DNA strand breaks caused by a DNA topoisomerase I poison in conjunction with DNA replication and RNA transcription

ABSTRACT

Camptothecin (CPT) inhibits DNA topoisomerase I (Top1) through a non-catalytic mechanism that stabilizes the Top1-DNA cleavage complex (Top1cc) and blocks the DNA re-ligation step, resulting in the accumulation in the genome of DNA single-strand breaks (SSBs), which are converted to secondary strand breaks when they collide with the DNA replication and RNA transcription machinery. DNA strand breaks mediated by replication, which have one DNA end, are distinct in repair from the DNA double-strand breaks (DSBs) that have two ends and are caused by ionizing radiation and other agents. In contrast to two-ended DSBs, such one-ended DSBs are preferentially repaired through the homologous recombination pathway. Conversely, the repair of one-ended DSBs by the non-homologous end-joining pathway is harmful for cells and leads to cell death. The choice of repair pathway has a crucial impact on cell fate and influences the efficacy of anticancer drugs such as CPT derivatives. In addition to replication-mediated one-ended DSBs, transcription also generates DNA strand breaks upon collision with the Top1cc. Some reports suggest that transcription-mediated DNA strand breaks correlate with neurodegenerative diseases. However, the details of the repair mechanisms of, and cellular responses to, transcription-mediated DNA strand breaks still remain unclear. In this review, combining our recent results and those of previous reports, we introduce and discuss the responses to CPT-induced DNA damage mediated by DNA replication and RNA transcription.

CELLULAR RESPONSES TO DNA DOUBLE-STRAND BREAKS

Cells are equipped with sophisticated surveillance and repair systems to guard against the deleterious effects of DNA damage. Central to this paradigm is a conserved network of proteins that transduces DNA damage signals to coordinate the processes of cell cycle arrest and DNA repair. The collapse of these systems renders cells vulnerable to genotoxic stresses and subsequently leads to genomic instability and an elevated susceptibility to cancer. Thus, many of the genes associated with the DNA damage response are tumor suppressor genes. On the other hand, the introduction of DNA damage into cancer cells via radiotherapy and/or cytotoxic chemotherapy has been a mainstay of cancer therapy for decades. Most of these cytotoxic agents kill cancer cells by causing DNA double-strand breaks (DSBs), and, as a consequence, there has been a major effort toward understanding how DSBs are recognized and repaired.

The two major DSB repair pathways are homologous recombination (HR) and non-homologous end joining (NHEJ) (O’Driscoll and Jeggo, 2006; Shibata and Jeggo, 2014) (Fig. 1), which are active in different stages of the cell cycle. HR is an error-free DSB repair mechanism that requires a sister chromatid and is therefore restricted to the S and G2 phases of the cell cycle. NHEJ is a fundamental mechanism to rejoin two DSB ends, and can occur throughout the cell cycle. Unlike HR, the NHEJ process is error-prone, often resulting in the introduction of mutations at the joining site (McVey and Lee, 2008; Shibata and Jeggo, 2014).

Fig. 1.

Two distinct types of DSBs leading to two distinct cell fates. DSBs induced by ionizing radiation are repaired through two pathways, HR and NHEJ, the latter being regulated and catalyzed by DNA-PKcs, Ku and DNA ligase IV (Lig4). Both HR and NHEJ are important for cells to survive DSBs. Replication-mediated one-ended DSBs induced by CPT are preferentially repaired through the HR pathway, resulting in cell survival; by contrast, NHEJ is toxic and results in cell death.

The molecular choreographies of HR and NHEJ have been elucidated and the following consensual model has been proposed. In NHEJ, the Ku complex, composed of Ku70 and Ku86 proteins, binds to the DNA end and recruits DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray repair cross-complementation group 4 (XRCC4) and DNA ligase IV. Other proteins, including Artemis and some polymerases, are also involved in NHEJ, depending on the situation (McVey and Lee, 2008; Lieber, 2010). On the other hand, in the initial steps of HR, the 5′ end is resected to expose the single-strand region for the strand exchange reaction; end resection is regulated by factors such as CtIP and the Mre11-Rad50-Nbs1 complex (Sartori et al., 2007; Takeda et al., 2007; Stracker and Petrini, 2011). The exposed single-stranded DNA is coated with replication protein A (RPA), followed by loading of Rad51, the protein responsible for strand exchange (O’Driscoll and Jeggo, 2006; San Filippo et al., 2008).

Phosphatidylinositol-3-kinase-related protein kinases including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs function as DNA damage sensors. ATM and DNA-PKcs are primarily activated in response to DSBs, whereas ATR functions as a major sensing factor in response to DNA replication stress (Cimprich and Cortez, 2008; Lovejoy and Cortez, 2009). ATM is immediately activated by DSB formation and contributes to the HR pathway by promoting chromatin remodeling and the DNA end resection required for subsequent strand exchange (Goodarzi et al., 2008; You et al., 2009), whereas DNA-PKcs senses DSBs as a complex with Ku proteins and promotes synapsis of two DNA ends and the subsequent end-joining reaction (DeFazio et al., 2002; Spagnolo et al., 2006).

How is the repair pathway for DSBs chosen? This is an open question, and many aspects of this process remain to be discovered. HR is suppressed by p53 binding protein 1 (53BP1), a large DNA damage response protein that accumulates around the DSB site and blocks DNA end resection (Iwabuchi et al., 1998; Rappold et al., 2001; Bothmer et al., 2010; Bunting et al., 2010). 53BP1 is recruited to the DSB site in response to the ubiquitination of histones and chromatin proteins by RING finger protein 8 (RNF8) and RNF168, E3 ubiquitin ligases that are involved in the DNA damage response; conversely, 53BP1 is excluded from the DSB site in a breast cancer-associated gene 1 (BRCA1)-dependent manner (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007; Doil et al., 2009; Stewart et al., 2009; Chapman et al., 2012; Kakarougkas et al., 2013). Thus, BRCA1-deficient cells cannot ensure DNA end resection, and end resection is likely to be a key reaction to select HR at the pathway choice step.

MECHANISMS OF CPT-INDUCED DNA DAMAGE

CPT is a natural alkaloid isolated from Camptotheca acuminata, and is known as a major DNA topoisomerase 1 (Top1) poison because it specifically targets Top1 and compromises its function. Top1 resolves the torsional stress accompanying DNA replication and RNA transcription through a cycle of DNA nicking and re-ligation. Top1 cleaves DNA by generating a covalent bond between the 3′ end of DNA and tyrosine 723 at the C terminus of Top1, and is immediately released from DNA by re-ligation of the nick after topoisomerization (Pommier, 2006). CPT blocks the re-ligation step and stabilizes the Top1-DNA cleavage complex (Top1cc) with Top1 still bound to the 3′ end of the nick (also referred to as a single-strand break [SSB]) (Fig. 2). Thus, CPT is a structural inhibitor of Top1 but not a catalytic chemical inhibitor. Although CPT itself has low water solubility, hydrosoluble derivatives have been developed for clinical use as anticancer drugs. One of these derivatives, topotecan, directly binds to the Top1cc, whereas another water-soluble derivative, irinotecan (CPT-11), is a prodrug which is processed by carboxyesterase in vivo, and then becomes the active form SN-38 (Mathijssen et al., 2001; Pizzolato and Saltz, 2003; Pommier, 2006).

Fig. 2.

A DNA replication-mediated strand break caused by CPT. The DNA replication fork collides with a CPT-trapped Top1cc, and the site is converted into a one-ended DSB. In the proposed model, Ku binds to a one-ended DSB, and should be removed immediately; otherwise, Ku can become an obstacle in HR. A Ku-bound one-ended DSB is highly toxic, leading to lethal chromosomal aberrations via NHEJ.

CPT-trapped Top1ccs are processed and repaired by a subset of the base excision repair (BER) system. In a proposed model, X-ray repair cross-complementation group 1 (XRCC1) recruits tyrosyl-DNA phosphodiesterase 1 (TDP1) and polynucleotide kinase 3′-phosphatase (PNKP). TDP1 cuts the phospho-tyrosyl bond between Top1 and the DNA, and PNKP converts the 3′-P to 3′-OH for subsequent repair by DNA polymerase and ligase (Dexheimer et al., 2008). Top1 is known to be ubiquitinated by Cul3 or Cul4 E3 ubiquitin ligase complexes and degraded by the proteasome (Zhang et al., 2004; Kerzendorfer et al., 2010). Previous studies have provided a tentative model in which the residual peptide of Top1 is removed from DNA by TDP1 after degradation (Debethune et al., 2002; Interthal and Champoux, 2011). Interestingly, Top1 degradation is dependent on transcription (Desai et al., 2003); however, the molecular basis of how transcription contributes to Top1 degradation has not yet been revealed.

Early clues as to how CPT causes DSBs came from the finding that CPT-induced toxicity was blocked by inhibitors of DNA replication (Hsiang et al., 1985; Avemann et al., 1988). Subsequent studies showed that Top1ccs are barriers to DNA replication and that a collision between an active replication fork and a Top1cc leads to the production of DSBs that are responsible for CPT toxicity (Hsiang et al., 1989; Ryan et al., 1991). However, an important structural distinction exists between the DSBs arising from replication fork-Top1cc collisions and those occurring in non-replicating DNA as a result of radiation- or chemical-induced damage. The latter scenario exposes two free DNA ends and is thus appropriately referred to as a DSB, whereas the replication fork-Top1cc collision exposes a one-ended DSB (Figs. 1 and 2).

CPT AND REPLICATION-MEDIATED DNA STRAND BREAKAGE

The cellular responses to CPT-induced one-ended DSBs have been investigated in a range of model organisms, from yeast to humans; these studies revealed that one-ended DSBs can be preferentially repaired through the HR pathway, leading to the reestablishment of the DNA replication fork (Helleday, 2003). Conversely, cells deficient in NHEJ are remarkably resistant to CPT (Adachi et al., 2004; Hochegger et al., 2006; Sakasai et al., 2010a), suggesting that NHEJ renders cells highly sensitive to one-ended DSBs and that the repair of one-ended DSBs by NHEJ causes cytotoxic chromosomal aberrations. Two recent papers provided data that support this hypothesis, reporting that CPT induces chromosomal aberrations such as radial chromosomes in an NHEJ-dependent manner (Bunting et al., 2010; Eid et al., 2010). This implies that the balance of NHEJ and HR is a critical element to determine cell fate in response to one-ended DSBs.

In yeast, the Ku complex is a suppressor of DNA end resection. CTP1- (yeast ortholog of human CtIP) and MRE11-deleted cells have defective DNA end resection, which is rescued by the additional deletion of the Ku80 gene (Langerak et al., 2011). Petrini and colleagues isolated a ku mutant in yeast that separately affects NHEJ-associated and NHEJ-independent functions. This ku mutation does not have a significant effect on NHEJ activity, but, like deletion of Ku, it rescues CPT sensitivity in the mre11 mutant (Balestrini et al., 2013). The mutation reduces the affinity of the Ku complex for DNA ends, enabling another nuclease, Exo1, to resect DNA ends in an Mre11-independent manner, resulting in the acquisition of resistance to CPT. This raises the possibility that Ku removal from DNA ends is critical for cell survival against CPT.

In response to CPT, RPA is rapidly and efficiently phosphorylated following its recruitment onto the single-stranded DNA generated by DNA end resection (Dutta and Stillman, 1992; Shao et al., 1999; Wang et al., 2001; Sakasai et al., 2006), implying that one-ended DSB is an eligible target for DNA end resection. Thus, although the relationship between Ku protein and DNA end resection in human cells has not been elucidated, unknown proteins that promote Ku removal from DNA ends may be important to execute HR in response to one-ended DSBs (Fig. 2). This hypothesis is supported by the observations that suppression of end resection by CtIP knockdown enhanced DNA-PKcs activation induced by CPT but not by etoposide, which induces two-ended DSBs (Eid et al., 2010; our unpublished data). Since DNA-PKcs activation is dependent on Ku protein, these results imply that efficient Ku removal from one-ended DSB is dependent on end resection in human cells. Moreover, observation of Ku nuclear foci with a high-resolution microscope revealed that although Ku barely forms foci in response to CPT, CPT-induced Ku foci can be observed with an ATM inhibitor (Britton et al., 2013). Since ATM inhibition is believed to down-regulate end resection (You et al., 2009), the Ku foci data further support the above model in which Ku is immediately removed from one-ended DSBs via ATM-dependent end resection that excludes NHEJ and starts HR.

The ubiquitin-proteasome pathway may be involved in the step of NHEJ exclusion at one-ended DSBs. We have previously reported that treatment with a proteasome inhibitor suppresses DNA-PKcs activation caused by CPT, but not by ionizing or ultraviolet radiation (Sakasai et al., 2010b). This suggests that DNA-PKcs possesses a ubiquitin-dependent mechanism in its activation induced by one-ended DSBs.

53BP1 requires RNF8-dependent ubiquitination of chromatin-related proteins for its recruitment to a DSB site, and its focus formation is suppressed by proteasome inhibition (Mailand et al., 2007; Sakasai and Tibbetts, 2008). However, CPT-induced DNA-PKcs activation is not dependent on RNF8 or 53BP1 (Sakasai et al., 2010b). We are currently attempting to identify the ubiquitination factors involved in DNA-PKcs activation induced by one-ended DSBs. In cells knocked down for a certain E2 ubiquitin-conjugating enzyme, DNA-PKcs activation was suppressed in response to CPT, but not to a DSB-inducing drug. The E2 protein was also involved in the generation of NHEJ-dependent chromosomal aberrations (our unpublished data). Thus, this E2 protein is a candidate for a new factor that regulates NHEJ in response to one-ended DSBs. Because this E2 protein does not affect DNA end resection, it may play an antagonistic role in NHEJ exclusion by blocking Ku removal from one-ended DSBs.

Details of pathway choice at one-ended DSBs are also emerging from studies of the cellular responses to non-CPT-type anticancer drugs, poly(ADP-ribose) polymerase (PARP) inhibitors. Recently, it has been reported that HR-deficient cancer cells, such as cells with mutated BRCA1 or BRCA2, are highly sensitive to PARP inhibitors. The synthetic lethal interaction between BRCA mutations and PARP inhibitors has been exploited to treat selected subsets of breast and ovarian cancer patients (Bryant et al., 2005; Fong et al., 2009). Although PARP inhibitors do not directly induce DNA strand breaks (Gottipati et al., 2010), they - like CPT - cause chromosome aberrations in an NHEJ-dependent manner in BRCA1-deficient cells (Bunting et al., 2010; Patel et al., 2011). Saleh-Gohari et al. (2005) suggest that a spontaneously collapsed replication fork produces a one-ended DSB, raising the possibility that the synthetic lethality between PARP inhibitors and BRCA mutation is caused by one-ended DSBs associated with spontaneously collapsed replication forks. Moreover, PARP-deficient cells exhibit CPT hypersensitivity in an NHEJ-dependent manner (Hochegger et al., 2006), suggesting that PARP contributes to the promotion of the HR pathway by suppressing NHEJ at one-ended DSBs.

Altogether, the choice between NHEJ and HR represents a major decision affecting cell fate after formation of one-ended DSBs, and NHEJ exhibits cytotoxic effects in the repair of one-ended DSBs but not two-ended DSBs (Fig. 1). However, a key question is whether NHEJ is actively excluded to give priority to HR in response to one-ended DSBs. We will not be able to answer this question until we develop techniques to detect the NHEJ reaction, particularly Ku behavior at one-ended DSBs.

CPT AND TRANSCRIPTION-MEDIATED DNA STRAND BREAKAGE

The bulk of research on CPT-induced responses has focused on replication-dependent DNA damage. However, the Top1cc represses RNA transcription as well as DNA replication and it is likely that collisions of the transcription machinery with Top1ccs contribute significantly to CTP-induced DNA damage even in non-dividing cells. Although DNA replication-mediated one-ended DSBs are cytotoxic, the cytotoxicity of transcription-mediated DNA strand breaks is not nearly as well understood. However, it has been reported that XRCC1-deficient cells still exhibit CPT sensitivity even when DNA replication is suppressed by aphidicolin treatment (Plo et al., 2003). This suggests that replication-independent cell death is caused by CPT in XRCC1-deficient cells and that transcriptional collapse through collision with the Top1cc leads to cytotoxicity.

The relationship between transcription and CPT-induced DNA damage was further illuminated through the investigation of 53BP1 foci in CPT-treated cells (Sakasai et al., 2010a). CPT induces two types of 53BP1 foci: uncountable fine foci in a pan-nuclear pattern, and much larger foci that typically range from 5–20 per cell. Small 53BP1 foci were mostly restricted to cells during S phase, whereas large 53BP1 foci were observed in G1 phase cells. Importantly, the appearance of large 53BP1 foci in G1 was blocked by transcription inhibitors, suggesting that they arise through collisions of the transcription machinery with Top1ccs. The transcription-dependent 53BP1 foci resemble DSB-like responses in two ways: they co-localize with phosphorylated histone H2AX (γH2AX), a major marker of DNA damage, and they depend on ATM and RNF8 for their formation. This suggests that the DNA damage response governed by ATM is activated by the collision between a Top1cc and the transcription machinery.

The DNA damage generated by collision between the transcription apparatus and a Top1cc is most likely DNA strand breaks, which may arise from a transcriptional intermediate that contains an RNA molecule, such as an R-loop, in a DNA-RNA hybrid structure (Fig. 3). Consistent with this idea, RNase H overexpression reduces transcription-dependent γH2AX focus formation (Sordet et al., 2009). DNA-PKcs activation is not observed in response to transcription-mediated DNA strand breaks (Sakasai et al., 2010a), which is consistent with the R-loop-like model, because Ku has a lower affinity for the DNA-RNA hybrid molecule (Mimori and Hardin, 1986).

Fig. 3.

An RNA transcription-mediated DNA strand break caused by CPT. The collision between a Top1cc and the transcription machinery results in DNA damage that potentially generates a transcription-mediated DNA strand break, which elicits DNA damage responses governed by ATM. Transcription-mediated DNA strand breaks are rapidly repaired by an unknown mechanism. Some genes responsible for neurodegenerative diseases, such as AT and CS, are involved in the response to transcription-mediated DNA strand breaks, suggesting an association between neurotoxicity and transcription-mediated DNA damage.

Replication- and transcription-dependent 53BP1 foci exhibit different kinetics of formation and disappearance. A time-course analysis revealed that replication-dependent foci occur within several minutes after CPT exposure, whereas transcription-dependent foci are only detectable after ~20 minutes. Focus disappearance exhibits the opposite pattern: replication-dependent foci remain for at least 4 hours after CPT washout, whereas transcription-dependent foci disappear within 2 hours (Sakai et al., 2012). These results suggest that replication-mediated one-ended DSBs rapidly activate the DNA damage response and require several hours for repair, whereas transcription-dependent foci have slower formation kinetics but the damage is repaired more quickly.

To investigate the cellular response to transcription-mediated DNA strand breaks, we have tried to identify factors that affect the formation of transcription-dependent 53BP1 foci. In PARP-inhibited cells, the number of 53BP1 foci was elevated (Sakai et al., 2012). One hypothesis is that the dysfunctional BER in PARP-inhibited cells allows the accumulation of Top1ccs and leads to an increase in 53BP1 focus number; however, the Top1cc level is not elevated in PARP-inhibited cells (Zhang et al., 2011). Knockdown of other BER factors, including XRCC1 and TDP1, did not show significant effects on 53BP1 focus formation, unlike those observed under PARP inhibition (Sakai et al., 2012). On the other hand, Zhang et al. (2011) reported that the number of CPT-induced γH2AX foci was increased in TDP1-knockout cells. They also demonstrated that elevated γH2AX focus formation under PARP inhibition was abolished by knockdown of the endonuclease XPF-ERCC1 (xeroderma pigmentosum [XP] complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1). This suggests that XP-related nucleases are involved in the generation of transcription-mediated strand breaks under the PARP-inactivated condition.

In contrast to PARP inhibiton, cells knocked down for CSB, the gene responsible for Cockayne syndrome (CS), have fewer foci and slower kinetics of focus formation than control cells. CSB is involved in transcription-coupled nucleotide excision repair, in which CSB recognizes stalled transcription machinery and promotes the subsequent recruitment of DNA repair proteins (Hanawalt and Spivak, 2008). Interestingly, it has been reported that CSB-deficient cells are sensitive to CPT (Squires et al., 1993). The molecular mechanism of their response to transcription-mediated DNA strand breaks is still under investigation; however, CSB may promote the recruitment of repair factors by recognizing collapsed transcriptional intermediates.

Transcription-mediated DNA strand breaks can be generated even in post-mitotic cells such as neurons. Neurodegenerative diseases and the DNA damage response are closely related. Ataxia telangiectasia (AT), XP and CS are diseases that involve both neurodegeneration and a deficiency in DNA damage response and repair. In neuronal cells deficient in ATM, the gene defective in AT, spontaneously and CPT-induced Top1ccs accumulate (Alagoz et al., 2013; Carlessi et al., 2014; Katyal et al., 2014), and Top1 degradation is down-regulated (Katyal et al., 2014). The relationship between transcription-dependent 53BP1 focus formation and Top1 degradation has not been elucidated; however, these observations suggest that ATM is critical for processing Top1ccs and Top1cc-derived secondary DNA damage, such as transcription-mediated DNA strand breaks. Furthermore, in evaluating the effectiveness of CPT as an anticancer drug, it may be important to consider Top1 degradation and the factors that regulate it because defective Top1 degradation is correlated with CPT sensitivity (Desai et al., 2001). Although CPT cytotoxicity is mainly derived from replication collapse, CPT-induced responses related to transcription may also contribute to anticancer efficacy.

SUMMARY

In contrast to two-ended DSBs, the repair pathway choice between HR and NHEJ for DNA replication-mediated one-ended DSBs has a crucial effect on cell fate. A method that promoted NHEJ could lead to more effective cancer chemotherapy. In addition, if site-specific induction of one-ended DSBs were possible, it would enable analysis of the detailed molecular behavior of DNA damage-response factors at one-ended DSBs, as well as research on the two-ended DSB response, in which site-specific endonucleases such as I-SceI have already contributed to important findings.

Elucidating the mechanism underlying transcription-mediated DNA strand breaks remains an important direction for future research. Induction of DNA strand breaks via transcription has been demonstrated in previous reports, but details such as the structure of DNA strand breaks and the identities of recognition and repair factors have not yet been elucidated. Further investigation of transcription-mediated DNA strand breaks may expand into the field of RNA metabolism, whose involvement in these processes has not yet been explored. Moreover, transcription-mediated DNA strand breaks influence post-mitotic cells such as neuronal cells. Indeed, TDP1 has been identified as an important gene in neurodegenerative disease (Takashima et al., 2002). Thus, the DNA damage response caused by CPT probably reflects one aspect of the underlying mechanism of neurodegenerative diseases. CPT is still the most promising tool for researching DNA damage induced by the collision between SSBs and DNA replication or RNA transcription, because no other available reagent with a similarly high versatility can specifically induce SSBs in the genome; moreover, research on CPT will continue to provide findings that are useful and applicable to effective cancer chemotherapy.

ACKNOWLEDGMENTS

We are grateful to T. Matsui, Y. Sunatani and R. S. Tibbetts for their critical reading of the manuscript.

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