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Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination



We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG.


Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines.


The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase.


CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.

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The authors are grateful to Drs. Asha Multani and Jin Ma (T. C. Hsu Molecular Cytogenetics Core at MDACC) for their technical support in cytogenetic studies (SCE).


This work was supported in part by grants R01 CA28596 (W. Plunkett), P50 CA100632 (W. Plunkett), and Cancer Center Support Grant P30 CA16672 from the National Cancer Institute, Department of Health and Human Services.

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XL and WP: conception and design. XL and YJ: development of methodology. XL, YJ, BN, SI, MO, and AM: acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.). XL, YJ, and WP: analysis and interpretation of data (e.g., statistical analysis, biostatistics, and computational analysis). XL, YJ, and WP: writing, review, and/or revision of the manuscript. XL, YJ, and BN: administrative, technical, or material support (i.e., reporting or organizing data, and constructing databases). WP: study supervision.

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Correspondence to William Plunkett.

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Supplemental Figure 1. Chromosomal aberrations induced by CNDAG 6-NH2. Representative images of mitotic spreads of AA8 and UV41 cells untreated and treated with 8 µM CNDAG 6-NH2 for 15 hours and 30 hours, respectively. Arrows indicate chromosomal breaks or gaps


Supplemental Figure 2. Cell cycle progression of CCRF-CEM cells after treatment with CNDAC or CNDAG derivatives. Suspension cells were exposed to 0.5 μM CNDAC, 50 μM CNDAG 6-OMe, and 50 μM CNDAG 6- NH2, respectively, for 24 hr. Cells were harvested, fixed and subjected to analysis by flow cytometry. (A) DNA content profiles are presented for all samples. (B) A stacked-bar plot is shown to present the percentage of all cell cycle phases in the whole populations


Supplemental Figure 3. Lack of p53 does not sensitize HCT116 cells to CNDAG. HCT116 cells with wild-type p53 (■) and p53 knocked-out (o) were washed into drugfree medium after a 24-hour exposure to CNDAG 6-NH2 at a range of concentrations and allowed to form colonies in 7–8 days. All points, mean ± SD of triplicate plates


Supplemental Figure 4. Sister chromatid exchanges in AA8 cells treated with mitomycin C (MMC). A representative image of metaphase chromosome spreads is shown.


Supplemental Figure 5. Proposed model for roles of DNA repair proteins in cellular response to CNDAG. CNDAG prodrugs are metabolized into the active form CNDAG under the action of adenosine deaminase (ADA). Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) are responsible for phosphorylating CNDAG. CNDAG triphosphates are incorporated into DNA and subsequently induce SSBs through β-elimination during the first S phase. The transcription-coupled nucleotide excision repair pathway (TC-NER) recognizes SSBs and contributes to cell survival (Wang, Liu, Matsuda, and Plunkett, 2008). Unrepaired SSBs are converted into DSBs mainly by replication in the second S phase. DSBs are lethal if left unrepaired. The homologous recombination (HR) pathway, which is activated by ATM, and involves RAD51, XRCC3 and BRCA2, is a major contributor to this survival function. The non-homologous end-joining (NHEJ) pathway is dispensable for cell survival of CNDAC-induced damage.

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Liu, X., Jiang, Y., Nowak, B. et al. Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination. Cancer Chemother Pharmacol 85, 661–672 (2020).

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  • Nucleoside analogue
  • Homologous recombination
  • ATM signaling pathway
  • XPF
  • T-ALL