Journal of Nucleic Acids

Journal of Nucleic Acids / 2010 / Article
Special Issue

DNA Damage, Mutagenesis, and DNA Repair

View this Special Issue

Review Article | Open Access

Volume 2010 |Article ID 543531 |

Natsuko Kondo, Akihisa Takahashi, Koji Ono, Takeo Ohnishi, "DNA Damage Induced by Alkylating Agents and Repair Pathways", Journal of Nucleic Acids, vol. 2010, Article ID 543531, 7 pages, 2010.

DNA Damage Induced by Alkylating Agents and Repair Pathways

Academic Editor: Ashis Basu
Received08 Jun 2010
Revised26 Aug 2010
Accepted12 Oct 2010
Published21 Nov 2010


The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at - and -atoms. -methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). -methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by -methylguanine-DNA methyltransferase, and MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine.

1. Introduction

Alkylating drugs are the oldest class of anticancer drugs still commonly used; they play an important role in the treatment of several types of cancers [1]. Most alkylating drugs are monofunctional methylating agents (e.g., temozolomide [TMZ], -methyl- -nitro- -nitrosoguanidine [MNNG], and dacarbazine), bifunctional alkylating agents such as nitrogen mustards (e.g., chlorambucil and cyclophosphamide), or chloroethylating agents (e.g., nimustine [ACNU], carmustine [BCNU], lomustine [CCNU], and fotemustine).

Simple methylating agents form adducts at the - and -atoms in DNA bases. -methylation adducts comprise more than 80% of methylated bases. These alkyl DNA base adducts exhibit different stabilities. For example, -methylguanine ( MeG) is the most stable -methylation adduct in vitro with a half-life ( ) no longer than 80 h [2]. Although -methylguanine ( MeG) accounts for only 0.3% (for methyl methanesulfonate) to 8% (for methylnitrosourea) of the total DNA methyl adducts, it is stable and persists in the absence of the DNA repair protein -methylguanine-DNA methyltransferase (MGMT) [35]. -methylthymine ( MeT) is produced at a much lower level (<0.4%) [2], and its mutagenicity and cytotoxicity are unclear. In general, -alkylations (e.g., alkylG and alkylT) are highly mutagenic and genotoxic, whereas -alkylations (e.g., alkylA and alkylA) are cytotoxic, but less mutagenic [69]. The primary products of methylating agents, -alkylated purines, are efficiently removed by base excision repair (BER) or human AlkB homologues (hABH). BER repairs MeG, MeA, and MeG, whereas hABH repairs MeA, MeC, MeT, and MeG [10].

One-step repair of MeG involves transferring the alkyl group from the oxygen in the guanine to a cysteine residue in the catalytic pocket of MGMT [10]. Nucleotide excision repair (NER) is an elaborate repair system that removes bulky lesions from DNA in 27-nt to 29-nt oligomers. Because it is also capable of removing nonbulky lesions such as apurinic/apyrimidinic sites and MeG residues, NER plays a backup role for other repair systems [11]. Mismatch repair (MMR) is also important in the repair of MeG. If it is left unrepaired, replication over the MeG results in an MeG:T mismatch or MeG:C ambiguous pair [12]. In the next round of replication, the MeG:T becomes an A:T transition mutation, or the MeG:C is replicated again as an MeG:C pair or becomes an MeG:T mismatch [13]. The MeG:T or MeG:C is recognized by the MutSα  complex (hMSH2 and hMSH6), which initiates MMR to create a gapped duplex by incision of the newly replicated strand [13]. If MeG remains in the template, a futile repair loop can eventually result in highly toxic double-strand breaks (DSBs), which are intermediates in apoptotic and DSB repair pathways [13]. Accordingly, DSB repair pathways are activated by methylating agents [14, 15].

Bifunctional alkylating agents, such as chlorambucil or BCNU, are commonly used anticancer drugs. DNA lesions produced by these agents require complex repair mechanisms. The primary chloroethyl adducts at G are repaired by MGMT, but the secondary interstrand cross-links (ICLs) require NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1 (XPF-ERCC1)) for incision, Fanconi anemia (FA) repair, and homologous recombination (HR) for complete repair [16].

This paper will focus on the repair pathways for MeG generated by methylating agents and those for ICLs generated by bifunctional alkylating agents. We will also briefly discuss other alkylation damage defense and processing functions (hABH and BER).

2. DNA Repair Mechanisms for DNA Damage Induced by Methylating Agents

2.1. MGMT (Figure 1(a))

MGMT repairs -alkylation adducts but irreversibly inactivates MGMT itself in the process. In the absence of active MGMT, MeG forms MeG/T mismatches during replication. Early studies demonstrated that MGMT-deficient cells unable to repair MeG damage were more sensitive to the effects of methylating agents than normal cells expressing MGMT [17]. This observation has been utilized experimentally and clinically to target cells with an MGMT inhibitor, the MeG analogue benzylG [18]. However, in some tumors, dysfunction suppresses MGMT expression [19, 20] or hypermethylation of the MGMT promoter results in gene silencing [21]. The low basal MGMT activity makes these cells less vulnerable to the effects of benzylG. Kaina et al. reported that about 5% of all solid tumors assayed in their laboratory were completely deficient in MGMT [10]. In particular, 17% to 30% of gliomas lack MGMT [22, 23]. Because drug efficacy is likely to be limited if only MGMT is targeted in these tumors, new molecular targets are being sought.

2.2. MMR (Figure 1(b))

The cytotoxicity of monofunctional alkylating agents requires a functional MMR in the target cells. In fact, mammalian cells proficient in MMR are generally about 100-fold more sensitive to alkylating agents than their MMR-deficient counterparts [24, 25]. In MMR-deficient cells, DNA damage accumulates but does not trigger cell death. Thus, resistance to these cytotoxic agents is associated with loss of MMR activity, particularly in the absence of MGMT [26, 27]. The mechanism of action of monofunctional alkylating agents has been studied in cell lines and mouse models; results indicate that replication over unrepaired MeG:C results in an MeG:T mismatch (or possibly an MeG:C ambiguous pair). In the next round of replication, an MeG:T mismatch becomes an A:T transition mutation. An MeG:T or MeG:C pair is recognized by the MutSα complex, which initiates MMR. MMR creates a gapped duplex after incision of the newly replicated strand. The mere presence of MeG in the genomic DNA of MMR-proficient cells is not cytotoxic, even if the cells are allowed to undergo a round of replication during which MeG:C and MeG:T pairs form. To activate the G2/M DNA damage checkpoint, these mispairs must be recognized and processed. Cells treated with MNNG are not arrested in the first G2/M checkpoint, but G2/M arrest is commonly observed in the second cell cycle [28].

2.3. DSB Repair (Figure 1(c))

Although alkylating agents do not directly induce DSBs, DSBs are detected in wild-type cells and other cell culture systems after the processing of DNA lesions induced by alkylating agents [14, 15, 29, 30]. DSBs lead to cell death; therefore, cells defective in DSB repair are thought to be more sensitive to alkylating agents. Consistent with this hypothesis, studies have reported that DSB repair pathways are involved in the repair of DNA damage induced by alkylating agents [14, 15, 29].

DSBs are repaired through the HR and nonhomologous end joining (NHEJ) pathways [31]. In human cells, HR proteins include members of the MRN complex, which consist of meiotic recombination 11 (MRE11)/radiation-sensitive mutant 50 (Rad50)/Nijmegen breakage syndrome 1 (NBS1) as well as Rad51, the Rad51 paralogs (Rad51B, Rad51C, Rad51D, X-ray repair cross-complementing group 2 (XRCC2), and XRCC3), Rad54, and Rad54B [31]. Proteins involved in the NHEJ pathway include Ku70/80, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), ligase IV (Lig4), XRCC4 and Artemis [31].

HR, which is a generally error-free pathway, uses DNA homology to direct DNA repair; an undamaged chromatid serves as template for the repair of a broken sister chromatid. The products of the breast cancer susceptibility genes, BRCA1 and BRCA2 (also known as FA complementation group D1 or FANCD1), are also involved in the HR pathway [32].

HR is required for MNNG-treated cells to transition into the second cell cycle. Most mammalian cells that undergo cell cycle arrest after the second S-phase die; however, the surviving cells show a high frequency of sister chromatid exchanges (SCEs), indicative of DSB repair at collapsed replication forks [33]. Roos et al. reported that BRCA2/XRCC2-dependent HR, but not NHEJ, protects against MeG-triggered DSBs and chromosomal aberrations, leading to SCEs [14].

NHEJ, which is the simplest way to repair a DSB, involves the religation of broken DNA ends without a template; this type of repair does not preserve the original genetic information. NHEJ eliminates DSBs during the G1 phase of the cell cycle, when the lack of sister chromatids prevents HR [34].

Results of a clonogenic survival study showed that Lig4 plays a more important role in the repair of TMZ-induced DSBs than XRCC2 or Rad54 [15]. DSBs, which may arise from adducts other than MeG, such as TMZ-induced -methylpurines, are repaired within 24 h in Lig4-proficient cells. In contrast, up to 80% of the DSBs in Lig4-/- cells were not repaired [15]. In A172 glioblastoma cells, siRNA silencing of Lig4 increased cellular sensitivity to TMZ. After TMZ treatments, A172 cells with silenced Lig4 exhibited a 62.5% decrease in survival compared with control A172 cells; thus, modulating Lig4 activity may enhance tumor sensitivity to TMZ [15].

2.4. BER (Figure 1(d))

The alkylation adducts MeG, MeA, and MeG are repaired by the BER system, the main DNA repair system in mammalian cells used to eliminate small DNA base lesions [16]. Damaged bases are removed by a lesion-specific DNA glycosylase, in this case alkyladenine DNA glycosylase (Aag). The resulting abasic site is recognized by an apurinic/apyrimidinic endonuclease, APE1, which incises the damaged strand, leaving -OH and deoxyribose phosphate ( -dRP) groups at the margins. A DNA polymerase β- (pol-β-)mediated DNA synthesis step fills the single nucleotide gap [35, 36] and removes the cytotoxic -dRP group [37, 38]. Alternatively, DNA polymerase (pol-λ) or DNA polymerase-ι (pol-ι), both of which possess -dRP lyase activity, may participate in BER to remove this toxic repair intermediate [3941]. Finally, DNA ligase I, or a complex of DNA ligase III and XRCC1, conducts the final, nick-sealing step in the pathway [42].

In the absence of pol-β, cells cannot repair the BER intermediate dRP and are thus hypersensitive to the alkylating agent methyl methanesulfonate [37, 38, 43, 44]. For example, fibroblasts from a pol-β-null mutant mouse are highly sensitive to monofunctional alkylating agents, but not to BCNU [45]. Similarly, RNA interference-mediated pol-β suppression boosts TMZ efficacy, although a deficiency in pol-ι or pol-λ does not increase TMZ-mediated cytotoxicity [46]. Furthermore, loss of pol-β coupled with TMZ treatment triggers H2AX phosphorylation, indicating activation of the DNA damage response pathway by unrepaired lesions [46]. H2AX is a histone protein that is rapidly phosphorylated on Ser139 (γH2AX) when DNA breaks are introduced in mammalian cells following external damage and replication fork collapse [47, 48]. Poly(ADP-ribose) polymerase-1 (PARP-1) is activated by strand breaks and participates in gap sealing with DNA ligase III and XRCC1, but deficiencies in the subsequent steps of BER increase sensitivity to alkylating agents. Inhibition of PARP-1 by the inhibitor AG14361 restores sensitivity to TMZ in MMR-deficient cells that have lost killing sensitivity to MeG via the MGMT/MMR pathway [49]. The combination of TMZ with PARP-1 inhibitors is currently under investigation in several Phase I-II clinical trials.

2.5. Direct Reversal of Alkylation Damage by AlkB Homologues (Figure 1(e))

The E. coli protein AlkB is an oxidative DNA demethylase that repairs the cytotoxic lesions MeA and MeC. A detailed mapping of the human genome has identified eight hABH homologues. ABH2 and ABH3 belong to the alpha-ketoglutarate- and Fe(II)-dependent dioxygenase superfamily. These proteins repair MeA, MeC, MeT, and MeG by oxidative demethylation [50, 51]. Although hABH2 preferentially repairs double-stranded DNA, hABH3 acts more efficiently on single-stranded nucleic acids. Accordingly, hABH2 relocates to replication foci during S-phase, which suggested that hABH2 repairs DNA close to replication forks, whereas hABH3 maintains nuclear single-stranded DNA and RNA, potentially targeting genes undergoing transcription.

Bifunctional alkylating agents (e.g., nitrogen mustards (melphalan, chlorambucil, cyclophosphamide, and ifosfamide) and chloroethylnitrosoureas (BCNU and CCNU)) possess two reactive sites. These agents cross-link DNA with proteins or, alternatively, cross-link two DNA bases within the same DNA strand (intrastrand cross-links) or on opposite DNA strands (ICLs). ICLs, which block replication forks, are the most serious cytotoxic lesions produced by most bifunctional drugs. Accordingly, the extent of ICLs correlates well with the cytotoxicity of nitrogen mustard drugs [52].

Nitrogen mustards form cross-links, and chloroethylnitrosoureas form cross-links [53]. The chloroethylated of the cross-link can be repaired by MGMT; however, this adduct is unstable and undergoes intramolecular rearrangement producing an intermediary - -ethanoG. The - -ethanoG adduct may react with cytosine in the complementary strand to yield a highly toxic ICL between position 1 in the guanine residue and position 3 in the cytosine residue (1-(3-cytosinyl)-2-(1-guanosinyl)-ethane) [53].

ICL repair mechanisms are complex; therefore, they are only briefly summarized here. An ICL represents a formidable block to the DNA replication machinery and is unique in requiring a combination of FA repair, NER, translesion synthesis (TLS), and HR repair for efficient repair [54]. Although the FA pathway was initially characterized in terms of DNA cross-link repair, this pathway is also involved in homologous recombination and resolution of the replication arrest [55, 56]. Thirteen FA genes have been identified [54], although the precise function of many of these FA proteins is unclear. The FA core complex, which consists of eight FA proteins, is activated by DNA damage. Specifically, the FA proteins FANCM and FANCA Associating Polypeptide 24 form a heterodimer that binds DNA [57, 58] and appears to be involved in sensing DNA replication forks blocked at cross-links. The NER proteins ERCC1 and XPF make incisions on either side of the cross-link to generate a gap. The gap is then filled by translesion synthesis (TLS) polymerases ζ (Rev3 and Rev7 subunits) and Rev1 (part of the Rev3-Rev7 complex [59]). The FA core complex monoubiquitinates FANCD2 and its paralog FANCI, and the ubiquitinated FANCD2 then interacts with FANCD1 to promote HR [54].

Incision at the ICL could occur before or after lesion bypass, leaving a DSB subject to HR or NHEJ [60]. As expected, XRCC2 and Rad54 are involved in the repair of ACNU-induced DSBs, but surprisingly Lig4 plays the most important role in this process [29]. In Lig4-/- cells, levels of phosphorylated histone γH2AX increased more than 4-fold at 12 h and 6-fold at 24 h after ACNU treatment compared to its initial levels. In contrast, γH2AX levels were not markedly altered by ACNU in normal cells. In addition, ACNU treatment markedly reduced the colony-forming ability of A172 glioblastoma cells transfected with siRNA against Lig4 or XRCC2 compared to controls [29]. However, Lig4 siRNA rendered cells more sensitive to the effects of ACNU than did XRCC2 siRNA [29]. These data suggest NHEJ may also be involved in removing DSBs formed by unrepaired ICLs.

4. Conclusion

DNA repair pathways attenuate the therapeutic effects of alkylating agents; therefore, characterization of the repair pathways is essential for developing new treatments. For example, MGMT promoter hypermethylation results in gene silencing and therefore decreased MGMT activity; therefore, MGMT promoter hypermethylation may be a useful way to enhance the therapeutic efficacy of TMZ [61, 62].

Currently, clinical trials are testing DNA repair inhibitors that target PARP, BER, or MGMT in combination with alkylating agents [63]. In the case of benzylG, a phase I clinical trial has defined the maximum tolerated dose of a single dose of TMZ when combined with benzylG and has determined the dose of benzylG that depletes tumor MGMT activity for 48 h [64]. In addition, when combined with cytotoxic chemotherapy, myelosuppression appears to be significantly enhanced by benzylG, significantly reducing the required doses of alkylating agents [65]. The success of such approaches will depend on selective targeting of the tumor. Locoregional chemotherapy has recently been shown to improve the survival of glioma patients [66]. Therefore, combining a locoregional delivery system with the simultaneous downregulation of DNA repair pathways may decrease the amount of alkylating agent needed for chemotherapy, thereby reducing the severe side effects. In addition, new inhibitors against specific repair proteins, such as pol-β, BRCA2, or Lig4, should be developed because resistance against currently available inhibitors may develop.


This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also funded in part by a grant from the Central Research Institute of the Electric Power Industry in Japan and by a grant for Exploratory Research for Space Utilization from the Japan Space Forum.


  1. S. G. Chaney and A. Sancar, “DNA repair: enzymatic mechanisms and relevance to drug response,” Journal of the National Cancer Institute, vol. 88, no. 19, pp. 1346–1360, 1996. View at: Google Scholar
  2. D. T. Beranek, “Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents,” Mutation Research, vol. 231, no. 1, pp. 11–30, 1990. View at: Google Scholar
  3. R. Goth and M. F. Rajewsky, “Persistence of O6 ethylguanine in rat brain DNA: correlation with nervous system specific carcinogenesis by ethylnitrosourea,” Proceedings of the National Academy of Sciences of the United States of America, vol. 71, no. 3, pp. 639–643, 1974. View at: Google Scholar
  4. R. Goth-Goldstein, “Inability of Chinese hamster ovary cells to excise O6-alkylguanine,” Cancer Research, vol. 40, no. 7, pp. 2623–2624, 1980. View at: Google Scholar
  5. B. Kaina, A. A. van Zeeland, A. de Groot, and A. T. Natarajan, “DNA repair and chromosomal stability in the alkylating agent-hypersensitive Chinese hamster cell line 27-1,” Mutation Research, vol. 243, no. 3, pp. 219–224, 1990. View at: Google Scholar
  6. G. P. Margison, M. F. Santibáñez-Koref, and A. C. Povey, “Mechanisms of carcinogenicity/chemotherapy by O6-methylguanine,” Mutagenesis, vol. 17, no. 6, pp. 483–487, 2002. View at: Google Scholar
  7. M. Christmann, M. T. Tomicic, W. P. Roos, and B. Kaina, “Mechanisms of human DNA repair: an update,” Toxicology, vol. 193, no. 1-2, pp. 3–34, 2003. View at: Publisher Site | Google Scholar
  8. M. D. Wyatt, J. M. Allan, A. Y. Lau, T. E. Ellenberger, and L. D. Samson, “3-Methyladenine DNA glycosylases: structure, function, and biological importance,” BioEssays, vol. 21, no. 8, pp. 668–676, 1999. View at: Google Scholar
  9. H. E. Krokan, R. Standal, and G. Slupphaug, “DNA glycosylases in the base excision repair of DNA,” Biochemical Journal, vol. 325, no. 1, pp. 1–16, 1997. View at: Google Scholar
  10. B. Kaina, M. Christmann, S. Naumann, and W. P. Roos, “MGMT: key node in the battle against genotoxicity, carcinogenicity and apoptosis induced by alkylating agents,” DNA Repair, vol. 6, no. 8, pp. 1079–1099, 2007. View at: Publisher Site | Google Scholar
  11. A. Sancar, “Excision repair in mammalian cells,” The Journal of Biological Chemistry, vol. 270, no. 27, pp. 15915–15918, 1995. View at: Google Scholar
  12. H. T. Chen, A. Bhandoola, M. J. Difilippantonio et al., “Response to RAG-mediated V(D)J cleavage by NBS1 and γ-H2AX,” Science, vol. 290, no. 5498, pp. 1962–1964, 2000. View at: Publisher Site | Google Scholar
  13. G. P. Margison and M. F. Santibáñez-Koref, “O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy,” BioEssays, vol. 24, no. 3, pp. 255–266, 2002. View at: Publisher Site | Google Scholar
  14. W. P. Roos, T. Nikolova, S. Quiros et al., “Brca2/Xrcc2 dependent HR, but not NHEJ, is required for protection against O6-methylguanine triggered apoptosis, DSBs and chromosomal aberrations by a process leading to SCEs,” DNA Repair, vol. 8, no. 1, pp. 72–86, 2009. View at: Publisher Site | Google Scholar
  15. N. Kondo, A. Takahashi, E. Mori et al., “DNA ligase IV as a new molecular target for temozolomide,” Biochemical and Biophysical Research Communications, vol. 387, no. 4, pp. 656–660, 2009. View at: Publisher Site | Google Scholar
  16. F. Drabløs, E. Feyzi, P. A. Aas et al., “Alkylation damage in DNA and RNA—repair mechanisms and medical significance,” DNA Repair, vol. 3, no. 11, pp. 1389–1407, 2004. View at: Publisher Site | Google Scholar
  17. R. S. Day III, C. H. J. Ziolkowski, and D. A. Scudiero, “Defective repair of alkylated DNA by human tumour and SV40-transformed human cell strains,” Nature, vol. 288, no. 5792, pp. 724–727, 1980. View at: Google Scholar
  18. N. Hosoya and K. Miyagawa, “Clinical importance of DNA repair inhibitors in cancer therapy,” Memo—Magazine of European Medical Oncology, vol. 2, no. 1, pp. 9–14, 2009. View at: Publisher Site | Google Scholar
  19. M. D. Blough, M. C. Zlatescu, and J. G. Cairncross, “O6-methylguanine-DNA methyltransferase regulation by p53 in astrocytic cells,” Cancer Research, vol. 67, no. 2, pp. 580–584, 2007. View at: Publisher Site | Google Scholar
  20. S. J. Russell, Y.-W. Ye, P. G. Waber, M. Shuford, S. C. Schold Jr., and P. D. Nisen, “p53 Mutations, O6-alkylguanine DNA alkyltransferase activity, and sensitivity to procarbazine in human brain tumors,” Cancer, vol. 75, no. 6, pp. 1339–1342, 1995. View at: Google Scholar
  21. M. Esteller, S. R. Hamilton, P. C. Burger, S. B. Baylin, and J. G. Herman, “Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia,” Cancer Research, vol. 59, no. 4, pp. 793–797, 1999. View at: Google Scholar
  22. I. Preuss, I. Eberhagen, S. Haas et al., “O6-methylguanine-DNA methyltransferase activity in breast and brain tumors,” International Journal of Cancer, vol. 61, no. 3, pp. 321–326, 1995. View at: Google Scholar
  23. N. P. Lees, K. L. Harrison, E. Hill, C. Nicholas Hall, A. C. Povey, and G. P. Margison, “Heterogeneity of O6-alkylguanine-DNA alkyltransferase activity in colorectal cancer: implications for treatment,” Oncology, vol. 63, no. 4, pp. 393–397, 2002. View at: Publisher Site | Google Scholar
  24. P. Karran, “Mechanisms of tolerance to DNA damaging therapeutic drugs,” Carcinogenesis, vol. 22, no. 12, pp. 1931–1937, 2001. View at: Google Scholar
  25. L. Stojic, R. Brun, and J. Jiricny, “Mismatch repair and DNA damage signalling,” DNA Repair, vol. 3, no. 8-9, pp. 1091–1101, 2004. View at: Publisher Site | Google Scholar
  26. P. Branch, G. Aquilina, M. Bignami, and P. Karran, “Defective mismatch binding and a mutator phenotype in cells tolerant to DNA damage,” Nature, vol. 362, no. 6421, pp. 652–654, 1993. View at: Publisher Site | Google Scholar
  27. A. Kat, W. G. Thilly, W.-H. Fang, M. J. Longley, G.-M. Li, and P. Modrich, “An alkylation-tolerant, mutator human cell line is deficient in strand- specific mismatch repair,” Proceedings of the National Academy of Sciences of the United States of America, vol. 90, no. 14, pp. 6424–6428, 1993. View at: Google Scholar
  28. S. Quiros, W. P. Roos, and B. Kaina, “Processing of O6-methylguanine into DNA double-strand breaks requires two rounds of replication whereas apoptosis is also induced in subsequent cell cycles,” Cell Cycle, vol. 9, no. 1, pp. 168–178, 2010. View at: Google Scholar
  29. N. Kondo, A. Takahashi, E. Mori et al., “DNA ligase IV is a potential molecular target in ACNU sensitivity,” Cancer Science, vol. 101, no. 8, pp. 1881–1885, 2010. View at: Publisher Site | Google Scholar
  30. S. C. Naumann, W. P. Roos, E. Jöst et al., “Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53,” British Journal of Cancer, vol. 100, no. 2, pp. 322–333, 2009. View at: Publisher Site | Google Scholar
  31. T. Ohnishi, E. Mori, and A. Takahashi, “DNA double-strand breaks: their production, recognition, and repair in eukaryotes,” Mutation Research, vol. 669, no. 1-2, pp. 8–12, 2009. View at: Publisher Site | Google Scholar
  32. A. A. Davies, J. Y. Masson, M. J. McIlwraith et al., “Role of BRCA2 in control of the RAD51 recombination and DNA repair protein,” Molecular Cell, vol. 7, no. 2, pp. 273–282, 2001. View at: Publisher Site | Google Scholar
  33. N. Mojas, M. Lopes, and J. Jiricny, “Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA,” Genes and Development, vol. 21, no. 24, pp. 3342–3355, 2007. View at: Publisher Site | Google Scholar
  34. T. Helleday, J. Lo, D. C. van Gent, and B. P. Engelward, “DNA double-strand break repair: from mechanistic understanding to cancer treatment,” DNA Repair, vol. 6, no. 7, pp. 923–935, 2007. View at: Publisher Site | Google Scholar
  35. R. W. Sobol and S. H. Wilson, “Mammalian DNA β-polymerase in base excision repair of alkylation damage,” Progress in Nucleic Acid Research and Molecular Biology, vol. 68, pp. 57–74, 2001. View at: Google Scholar
  36. S. H. Wilson, R. W. Sobol, W. A. Beard, J. K. Horton, R. Prasad, and B. J. Vande Berg, “DNA polymerase β and mammalian base excision repair,” Cold Spring Harbor Symposia on Quantitative Biology, vol. 65, pp. 143–155, 2000. View at: Google Scholar
  37. R. W. Sobol, J. K. Horton, R. Kühn et al., “Requirement of mammalian DNA polymerase-β in base-excision repair,” Nature, vol. 379, no. 6561, pp. 183–186, 1996. View at: Publisher Site | Google Scholar
  38. R. W. Sobol, R. Prasad, A. Evenski et al., “The lyase activity of the DNA repair protein β-polymerase protects from DNA-damage-induced cytotoxicity,” Nature, vol. 405, no. 6788, pp. 807–810, 2000. View at: Publisher Site | Google Scholar
  39. R. Prasad, K. Bebenek, E. Hou et al., “Localization of the deoxyribose phosphate lyase active site in human DNA polymerase β by controlled proteolysis,” The Journal of Biological Chemistry, vol. 278, no. 32, pp. 29649–29654, 2003. View at: Publisher Site | Google Scholar
  40. K. Bebenek, A. Tissier, E. G. Frank et al., “5-deoxyribose phosphate lyase activity of human DNA polymerase L in vitro,” Science, vol. 291, no. 5511, pp. 2156–2159, 2001. View at: Publisher Site | Google Scholar
  41. M. García-Díaz, K. Bebenek, T. A. Kunkel, and L. Blanco, “Identification of an intrinsic 5-deoxyribose-5-phosphate lyase activity in human DNA polymerase λ: a possible role in base excision repair,” The Journal of Biological Chemistry, vol. 276, no. 37, pp. 34659–34663, 2001. View at: Publisher Site | Google Scholar
  42. T. Lindahl and R. D. Wood, “Quality control by DNA repair,” Science, vol. 286, no. 5446, pp. 1897–1905, 1999. View at: Publisher Site | Google Scholar
  43. R. W. Sobolt, M. Kartalou, K. H. Almeida et al., “Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses,” The Journal of Biological Chemistry, vol. 278, no. 41, pp. 39951–39959, 2003. View at: Publisher Site | Google Scholar
  44. R. W. Sobol, D. E. Watson, J. Nakamura et al., “Mutations associated with base excision repair deficiency and methylation-induced genotoxic stress,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 10, pp. 6860–6865, 2002. View at: Publisher Site | Google Scholar
  45. J. K. Horton, D. F. Joyce-Gray, B. F. Pachkowski, J. A. Swenberg, and S. H. Wilson, “Hypersensitivity of DNA polymerase β null mouse fibroblasts reflects accumulation of cytotoxic repair intermediates from site-specific alkyl DNA lesions,” DNA Repair, vol. 2, no. 1, pp. 27–48, 2003. View at: Publisher Site | Google Scholar
  46. R. N. Trivedi, K. H. Almeida, J. L. Fornsaglio, S. Schamus, and R. W. Sobol, “The role of base excision repair in the sensitivity and resistance to temozolomide-mediated cell death,” Cancer Research, vol. 65, no. 14, pp. 6394–6400, 2005. View at: Publisher Site | Google Scholar
  47. E. P. Rogakou, D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner, “DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139,” The Journal of Biological Chemistry, vol. 273, no. 10, pp. 5858–5868, 1998. View at: Publisher Site | Google Scholar
  48. I. M. Ward and J. Chen, “Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress,” The Journal of Biological Chemistry, vol. 276, no. 51, pp. 47759–47762, 2001. View at: Google Scholar
  49. N. J. Curtin, L.-Z. Wang, A. Yiakouvaki et al., “Novel poly(ADP-ribose) polymerase-1 inhibitor, AG14361, restores rensitivity to temozolomide in mismatch repair-deficient cells,” Clinical Cancer Research, vol. 10, no. 3, pp. 881–889, 2004. View at: Publisher Site | Google Scholar
  50. T. Duncan, S. C. Trewick, P. Koivisto, P. A. Bates, T. Lindahl, and B. Sedgwick, “Reversal of DNA alkylation damage by two human dioxygenases,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 26, pp. 16660–16665, 2002. View at: Publisher Site | Google Scholar
  51. P. A. Aas, M. Otterlei, P. O. Falnes et al., “Human and bacterial oxidative demethylases repair alkylation damage in both RNA and DNA,” Nature, vol. 421, no. 6925, pp. 859–863, 2003. View at: Publisher Site | Google Scholar
  52. P. M. O'Connor and K. W. Kohn, “Comparative pharmacokinetics of DNA lesion formation and removal following treatment of L1210 cells with nitrogen mustards,” Cancer Communications, vol. 2, no. 12, pp. 387–394, 1990. View at: Google Scholar
  53. M. R. Middleton and G. P. Margison, “Improvement of chemotherapy efficacy by inactivation of a DNA-repair pathway,” Lancet Oncology, vol. 4, no. 1, pp. 37–44, 2003. View at: Publisher Site | Google Scholar
  54. L. H. Thompson and J. M. Hinz, “Cellular and molecular consequences of defective Fanconi anemia proteins in replication-coupled DNA repair: mechanistic insights,” Mutation Research, vol. 668, no. 1-2, pp. 54–72, 2009. View at: Publisher Site | Google Scholar
  55. K. Yamamoto, M. Ishiai, N. Matsushita et al., “Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells,” Molecular and Cellular Biology, vol. 23, no. 15, pp. 5421–5430, 2003. View at: Publisher Site | Google Scholar
  56. K. Nakanishi, Y.-G. Yang, A. J. Pierce et al., “Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 4, pp. 1110–1115, 2005. View at: Publisher Site | Google Scholar
  57. A. R. Meetei, A. L. Medhurst, C. Ling et al., “A human ortholog of archaeal DNA repair protein Hef is defective in Fanconi anemia complementation group M,” Nature Genetics, vol. 37, no. 9, pp. 958–963, 2005. View at: Publisher Site | Google Scholar
  58. A. Ciccia, C. Ling, R. Coulthard et al., “Identification of FAAP24, a Fanconi Anemia Core Complex Protein that Interacts with FANCM,” Molecular Cell, vol. 25, no. 3, pp. 331–343, 2007. View at: Publisher Site | Google Scholar
  59. Y. Masuda, M. Ohmae, K. Masuda, and K. Kamiya, “Structure and enzymatic properties of a stable complex of the human REV1 and REV7 proteins,” The Journal of Biological Chemistry, vol. 278, no. 14, pp. 12356–12360, 2003. View at: Publisher Site | Google Scholar
  60. M. L. G. Dronkert and R. Kanaar, “Repair of DNA interstrand cross-links,” Mutation Research, vol. 486, no. 4, pp. 217–247, 2001. View at: Publisher Site | Google Scholar
  61. M. E. Hegi, A.-C. Diserens, T. Gorlia et al., “MGMT gene silencing and benefit from temozolomide in glioblastoma,” The New England Journal of Medicine, vol. 352, no. 10, pp. 997–1003, 2005. View at: Publisher Site | Google Scholar
  62. M. Esteller, J. Garcia-Foncillas, E. Andion et al., “Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents,” The New England Journal of Medicine, vol. 343, no. 13, pp. 1350–1354, 2000. View at: Publisher Site | Google Scholar
  63. T. Helleday, E. Petermann, C. Lundin, B. Hodgson, and R. A. Sharma, “DNA repair pathways as targets for cancer therapy,” Nature Reviews Cancer, vol. 8, no. 3, pp. 193–204, 2008. View at: Publisher Site | Google Scholar
  64. J. A. Quinn, A. Desjardins, J. Weingart et al., “Phase I trial of temozolomide plus O6-benzylguanine for patients with recurrent or progressive malignant glioma,” Journal of Clinical Oncology, vol. 23, no. 28, pp. 7178–7187, 2005. View at: Publisher Site | Google Scholar
  65. O. Khan and M. R. Middleton, “The therapeutic potential of O6-alkylguanine DNA alkyltransferase inhibitors,” Expert Opinion on Investigational Drugs, vol. 16, no. 10, pp. 1573–1584, 2007. View at: Publisher Site | Google Scholar
  66. A. Giese, T. Kucinski, U. Knopp et al., “Pattern of recurrence following local chemotherapy with biodegradable carmustine (BCNU) implants in patients with glioblastoma,” Journal of Neuro-Oncology, vol. 66, no. 3, pp. 351–360, 2004. View at: Publisher Site | Google Scholar

Copyright © 2010 Natsuko Kondo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

More related articles

 PDF Download Citation Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles