Post-Integrational DNA Repair of HIV-1 Is Associated with the Activation of DNA-PK and ATM Cellular Protein Kinases and Phosphorylation of Their Targets

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Abstract

Integration of the DNA copy of the HIV-1 genome into the cellular genome results in series of damages, the repair of which is critical for successful viral replication. We have previously demonstrated that the ATM and DNA-PK kinases, normally responsible for repairing double-strand breaks in the cellular DNA, are required to initiate HIV-1 post-integration repair, even though integration does not result in double-strand DNA breaks. In this study, we analyzed changes in the phosphorylation status of ATM (pSer1981), DNA-PK (pSer2056) and their related kinase ATR (pSer428), as well as their targets: Chk1 (pSer345), Chk2 (pThr68), H2AX (pSer139) and p53 (pSer15) during HIV-1 post-integration repair. We have shown that ATM and DNA-PK, but not ATR, undergo autophosphorylation during postintegration DNA repair and phosphorylate their target proteins Chk2 and H2AX. These data indicate common signaling mechanisms between double-strand DNA break repair and postintegration repair of HIV-1.

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A. N. Anisenko

Lomonosov Moscow State University

Author for correspondence.
Email: a_anisenko@mail.ru
Russian Federation, Moscow

A. A. Nefedova

Lomonosov Moscow State University

Email: a_anisenko@mail.ru
Russian Federation, Moscow

I. I. Kireev

Lomonosov Moscow State University

Email: a_anisenko@mail.ru
Russian Federation, Moscow

M. B. Gottikh

Lomonosov Moscow State University

Email: a_anisenko@mail.ru
Russian Federation, Moscow

References

  1. Lesbats, P., Engelman, A. N., and Cherepanov, P. (2016) Retroviral DNA Integration, Chem. Rev., 116, 12730-12757, https://doi.org/10.1021/acs.chemrev.6b00125.
  2. Vincent, K. A., York-Higgins, D., Quiroga, M., and Brown, P. O. (1990) Host sequences flanking the HIV provirus, Nucleic Acids Res., 18, 6045-6047, https://doi.org/10.1093/nar/18.20.6045.
  3. Vink, C., Groenink, M., Elgersma, Y., Fouchier, R. A., Tersmette, M., and Plasterk, R. H. (1990) Analysis of the junctions between human immunodeficiency virus type 1 proviral DNA and human DNA, J. Virol., 64, 5626-5627, https://doi.org/10.1128/jvi.64.11.5626-5627.1990.
  4. Княжанская Е. С., Шадрина О. А., Анисенко А. Н., Готтих М. Б. (2016) Роль ДНК-зависимой протеинкиназы в репликации ВИЧ-1, Мол. Биол., 50, 639-654.
  5. Анисенко А. Н., Готтих М. Б. (2019) Участие клеточных систем репарации ДНК в репликации ВИЧ-1, Мол. Биол., 53, 355-366.
  6. Skalka, A. M., and Katz, R. A. (2005) Retroviral DNA integration and the DNA damage response, Cell Death Differ., 12, 971-978, https://doi.org/10.1038/sj.cdd.4401573.
  7. Daniel, R., Katz, R. A., and Skalka, A. M. (1999) A role for DNA-PK in retroviral DNA integration, Science, 284, 644-647, https://doi.org/10.1126/science.284.5414.644.
  8. Daniel, R., Greger, J. G., Katz, R. A., Taganov, K. D., Wu, X., Kappes, J. C., and Skalka, A. M. (2004) Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway, J. Virol., 78, 8573-8581, https://doi.org/10.1128/JVI.78.16.8573-8581.2004.
  9. Smith, J. A., Wang, F. X., Zhang, H., Wu, K. J., Williams, K. J., and Daniel, R. (2008) Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair, Virol. J., 5, 11, https://doi.org/10.1186/1743-422X-5-11.
  10. Lau, A., Swinbank, K. M., Ahmed, P. S., Taylor, D. L., Jackson, S. P., Smith, G. C. M., and O’Connor, M. J. (2005) Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase, Nat. Cell Biol., 7, 493-500, https://doi.org/10.1038/ncb1250.
  11. Knyazhanskaya, E., Anisenko, A., Shadrina, O., Kalinina, A., Zatsepin, T., Zalevsky, A., Mazurov, D., and Gottikh, M. (2019) NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase, Retrovirology, 16, 30, https://doi.org/10.1186/s12977-019-0492-z.
  12. Anisenko, A., Nefedova, A., Agapkina, Y., and Gottikh, M. (2023) Both ATM and DNA-PK are the main regulators of HIV-1 post-integrational DNA repair, Int. J. Mol. Sci., 24, 2797, https://doi.org/10.3390/ijms24032797.
  13. Blackford, A. N., and Jackson, S. P. (2017) ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response, Mol. Cell, 66, 801-817, https://doi.org/10.1016/j.molcel.2017.05.015.
  14. Anisenko, A. N., Knyazhanskaya, E. S., Zalevsky, A. O., Agapkina, J. Y., Sizov, A. I., Zatsepin, T. S., and Gottikh, M. B. (2017) Characterization of HIV-1 integrase interaction with human Ku70 protein and initial implications for drug targeting, Sci. Rep., 7, 5649, https://doi.org/10.1038/s41598-017-05659-5.
  15. Delelis, O., Carayon, K., Saïb, A., Deprez, E., and Mouscadet, J. F. (2008) Integrase and integration: biochemical activities of HIV-1 integrase, Retrovirology, 5, 114, https://doi.org/10.1186/1742-4690-5-114.
  16. Wu, X., Liu, H., Xiao, H., Conway, J. A., Hehl, E., Kalpana, G. V., Prasad, V., and Kappes, J. C. (1999) Human immunodeficiency virus type 1 integrase protein promotes reverse transcription through specific interactions with the nucleoprotein reverse transcription complex, J. Virol., 73, 2126-2135, https://doi.org/10.1128/JVI.73.3.2126-2135.1999.
  17. Stiff, T., O’Driscoll, M., Rief, N., Iwabuchi, K., Löbrich, M., and Jeggo, P A. (2004) ATM and DNA-PK function redundantly to phosphorylate H2AX after exposure to ionizing radiation, Cancer Res., 64, 2390-2396, https://doi.org/10.1158/0008-5472.CAN-03-3207.
  18. Rothkamm, K., and Löbrich, M. (2003) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses, Proc. Natl. Acad. Sci. USA, 100, 5057-5062, https://doi.org/10.1073/pnas.0830918100.
  19. Menolfi, D., and Zha, S. (2020) ATM, ATR and DNA-PKcs kinases – the lessons from the mouse models: inhibition ≠ deletion, Cell Biosci., 10, 8, https://doi.org/10.1186/s13578-020-0376-x.
  20. Schlam‐Babayov, S., Bensimon, A., Harel, M., Geiger, T., Aebersold, R., Ziv, Y., and Shiloh, Y. (2021) Phosphoproteomics reveals novel modes of function and inter‐relationships among PIKKs in response to genotoxic stress, EMBO J., 40, e104400, https://doi.org/10.15252/embj.2020104400.
  21. Anisenko, A., Kan, M., Shadrina, O., Brattseva, A., and Gottikh, M. (2020) Phosphorylation targets of DNA-PK and their role in HIV-1 replication, Cells, 9, 1908, https://doi.org/10.3390/cells9081907.
  22. Bensimon, A., Schmidt, A., Ziv, Y., Elkon, R., Wang, S. Y., Chen, D. J., Aebersold, R., and Shiloh, Y. (2010) ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage, Sci. Signal., 3, rs3, https://doi.org/10.1126/scisignal.2001034.
  23. Finzel, A., Grybowski, A., Strasen, J., Cristiano, E., and Loewer, A. (2016) Hyperactivation of ATM upon DNA-PKcs inhibition modulates p53 dynamics and cell fate in response to DNA damage, Mol. Biol. Cell, 27, 2360-2367, https://doi.org/10.1091/mbc.e16-01-0032.
  24. Schlam-Babayov, S., Ziv, Y., and Shiloh, Y. (2021) It takes three to the DNA damage response tango, Mol. Cell. Oncol., 8, 1881395, https://doi.org/10.1080/23723556.2021.1881395.
  25. Lu, H., Zhang, Q., Laverty, D. J., Puncheon, A. C., Augustine, M. M., Williams, G. J., Nagel, Z. D., Chen, B. P. C., and Davis, A. J. (2023) ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining, Nucleic Acids Res., 51, 6770-6783, https://doi.org/10.1093/nar/gkad505.
  26. Mladenov, E., Fan, X., Paul-Konietzko, K., Soni, A., and Iliakis, G. (2019) DNA-PKcs and ATM epistatically suppress DNA end resection and hyperactivation of ATR-dependent G2-checkpoint in S-phase irradiated cells, Sci. Rep., 9, 14597, https://doi.org/10.1038/s41598-019-51071-6.
  27. Liu, S., Opiyo, S. O., Manthey, K., Glanzer, J. G., Ashley, A. K., Amerin, C., Troksa, K., Shrivastav, M., Nickoloff, J. A., and Oakley, G. G. (2012) Distinct roles for DNA-PK, ATM and ATR in RPA phosphorylation and checkpoint activation in response to replication stress, Nucleic Acids Res., 40, 10780-10794, https://doi.org/10.1093/nar/gks849.
  28. Zhou, Y., Lee, J. H., Jiang, W., Crowe, J. L., Zha, S., and Paull, T. T. (2017) Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM, Mol. Cell, 65, 91-104, https://doi.org/10.1016/j.molcel.2016.11.004.
  29. Kurosawa, A. (2021) Autophosphorylation and self-activation of DNA-dependent protein kinase, Genes, 12, 1091, https://doi.org/10.3390/genes12071091.
  30. Hsu, F. M., Zhang, S., and Chen, B. P. C. (2012) Role of DNA-dependent protein kinase catalytic subunit in cancer development and treatment, Transl. Cancer Res., 1, 22-34, https://doi.org/10.3978/j.issn.2218-676X.2012.04.01.
  31. Lafont, F., Fleury, F., and Benhelli-Mokrani, H. (2020) DNA-PKcs Ser2056 auto-phosphorylation is affected by an O-GlcNAcylation/phosphorylation interplay, Biochim. Biophys. Acta Gen. Subj., 1864, 129705, https://doi.org/10.1016/j.bbagen.2020.129705.
  32. Chen, B. P. C., Chan, D. W., Kobayashi, J., Burma, S., Asaithamby, A., Morotomi-Yano, K., Botvinick, E., Qin, J., and Chen, D. J. (2005) Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks, J. Biol. Chem., 280, 14709-14715, https://doi.org/10.1074/jbc.M408827200.
  33. Bakkenist, C. J., and Kastan, M. B. (2003) DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation, Nature, 421, 499-506, https://doi.org/10.1038/nature01368.
  34. So, S., Davis, A. J., and Chen, D. J. (2009) Autophosphorylation at serine 1981 stabilizes ATM at DNA damage sites, J. Cell Biol., 187, 977-990, https://doi.org/10.1083/jcb.200906064.
  35. Yuan, Z., Guo, W., Yang, J., Li, L., Wang, M., Lei, Y., Wan, Y., Zhao, X., Luo, N., Cheng, P., Liu, X., Nie, C., Peng, Y., Tong, A., and Wei, Y. (2015) PNAS-4, an early DNA damage response gene, induces S phase arrest and apoptosis by activating checkpoint kinases in lung cancer cells, J. Biol. Chem., 290, 14927, https://doi.org/10.1074/jbc.M115.658419.
  36. Li, J., and Stern, D. F. (2005) Regulation of CHK2 by DNA-dependent protein kinase, J. Biol. Chem., 280, 12041-12050, https://doi.org/10.1074/jbc.M412445200.
  37. Boehme, K. A., Kulikov, R., and Blattner, C. (2008) p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK, Proc. Natl. Acad. Sci. USA, 105, 7785-7790, https://doi.org/10.1073/pnas.0703423105.
  38. Shieh, S. Y., Ikeda, M., Taya, Y., and Prives, C. (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2, Cell, 91, 325-334, https://doi.org/10.1016/S0092-8674(00)80416-X.
  39. Loughery, J., Cox, M., Smith, L. M., and Meek, D. W. (2014) Critical role for p53-serine 15 phosphorylation in stimulating transactivation at p53-responsive promoters, Nucleic Acids Res., 42, 7666-7680, https://doi.org/10.1093/nar/gku501.
  40. Podhorecka, M., Skladanowski, A., and Bozko, P. (2010) H2AX phosphorylation: its role in DNA damage response and cancer therapy, J. Nucleic Acids, 2010, 920161, https://doi.org/10.4061/2010/920161.
  41. Liu, S., Shiotani, B., Lahiri, M., Maréchal, A., Tse, A., Leung, C. C. Y., Glover, J. N. M., Yang, X. H., and Zou, L. (2011) ATR autophosphorylation as a molecular switch for checkpoint activation, Mol. Cell, 43, 192-202, https://doi.org/10.1016/j.molcel.2011.06.019.
  42. Kulkarni, A., and Das, K. C. (2008) Differential roles of ATR and ATM in p53, Chk1, and histone H2AX phosphorylation in response to hyperoxia: ATR-dependent ATM activation, Am. J. Physiol. Lung Cell. Mol. Physiol., 294, 998-1006, https://doi.org/10.1152/ajplung.00004.2008.

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2. Fig. 1. Analysis of protein phosphorylation levels of DNA-PKcs, ATM, ATR, Chk1, Chk2, p53 and H2AX in cells transduced with HIV_wt, HIV_mut or non-transduced control (Cntr), 12 h after addition of lentiviral vectors. The graph shows the levels of the phosphorylated form of the protein in cells transduced with HIV_wt or HIV_mut relative to the nontransduced sample (mean ± SD over three independent repeats). Statistical significance of changes in phosphorylated protein levels was assessed by two-factor ANOVA with Tukey's multiple comparisons correction; ns - no statistical difference, **** p-value < 0.0001

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3. Fig. 2. Western blot analysis of phosphorylated forms of p53 (pSer15) and H2AX (pSer139). Cntr, non-transduced cells transduced with vector; HIV_wt, HIV_mut, HIV_E152A, HIV_F185A, cells transduced with HIV-1-based vectors with the natural integrase variant or the corresponding mutant form; HIV_wt + Nu7441 and HIV_wt + Ku-55933 samples after transduction of cells with HIV_wt vector were additionally treated with DNA-PKcs inhibitor - Nu7441 (2 μM), or ATM inhibitor - Ku-55933 (5 μM).

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4. Fig. 3. Kinetics of γH2AX and pSer15-p53 accumulation in HEK293T cells transduced with HIV_wt, HIV_mut or HIV_E152A vectors at 10, 12, 14 and 18.5 h after transduction. a - Western blot analysis; b - quantitative analysis of Western blotting results. The level of γH2AX and pSer15-p53 in cells transduced with HIV_wt 18.5 h after vector addition was taken as 1. The sample labelled as Cntr was not transduced with the vector

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5. Fig. 4. Accumulation of γH2AX loci in HEK293T cells transduced with HIV-1 genome-based vectors. a-e - Confocal images of γH2AX loci in HEK293T cells untreated (a), treated with 50 μM etoposide for 1 h before fixation (b), transduced with HIV_wt (c), HIV_mut (d), HIV_E152A (e), HIV_F185A (f) vectors at a multiplicity of infection (MOI) of 10. Cell fixation was performed 12 h after transduction. g - Mean number of γH2AX loci per cell (a total of 5000 cells were analysed in three technical repeats for each exposure type); statistical significance of differences was assessed using analysis of variance (ANOVA) with Tukey's correction for multiple comparisons; ns - no differences, *** p-value < 0.001, **** p-value < 0.0001; h - mean fluorescence intensity of γH2AX loci; statistical significance of differences was assessed using t-test, ** p-value < 0.01

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6. Fig. 5. Phosphorylation of proteins from cellular response systems to double-stranded DNA breaks during HIV-1 post-integration repair. HIV-1 integrase, located at HIV-1 genome insertion sites and therefore labelling DNA damage, attracts the heterodimeric Ku70-Ku80 complex. This complex is strictly required for the recruitment of DNA-PK and ATM and the subsequent activation of these kinases at sites of DNA damage induced by HIV-1 integration. DNA-PK and ATM are autophosphorylated by pSer2056 and pSer1981. Kinases activated during PIR phosphorylate H2AX and Chk2 targets, but not p53. ATR is not involved in PIR and is not activated

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