Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide

  1. Robert P Fuchs  Is a corresponding author
  2. Asako Isogawa
  3. Joao A Paulo
  4. Kazumitsu Onizuka
  5. Tatsuro Takahashi
  6. Ravindra Amunugama
  7. Julien P Duxin
  8. Shingo Fujii
  1. INSERM / AMU, France
  2. CRCM Marseille, France
  3. Harvard Medical School, United States
  4. Tohoku Univ, Sendai, Japan
  5. Kyushu Univ, Fukuoka, Japan
  6. Harvard, BCMP, United States
  7. Copenhagen University, Denmark

Abstract

Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG). Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-Methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We show that in this system, mismatch repair (MMR) proteins are enriched on MNU-treated DNA and we observe robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increased linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving BER acting at a nearby N-alkylation adducts. We propose a new, replication-independent mechanism of action of TMZ, that operates in addition to the well-studied cell cycle dependent mode of action.

Data availability

Source data files have been provided for MS data, gels and blots in main or supplementary figures.

Article and author information

Author details

  1. Robert P Fuchs

    Marseille Medical Genetics UMR1251, INSERM / AMU, Marseille, France
    For correspondence
    robert.fuchs@inserm.fr
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1098-4325
  2. Asako Isogawa

    DNA Repair, CRCM Marseille, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.
  3. Joao A Paulo

    Department of Cell Biology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Kazumitsu Onizuka

    Institute of Multidisciplinary Research for Advanced Materials, Tohoku Univ, Sendai, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  5. Tatsuro Takahashi

    Biology, Kyushu Univ, Fukuoka, Fukuoka, Japan
    Competing interests
    The authors declare that no competing interests exist.
  6. Ravindra Amunugama

    BCMP, Harvard, BCMP, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Julien P Duxin

    Center for Protein Research, Copenhagen University, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  8. Shingo Fujii

    DNA Repair, CRCM Marseille, Marseille, France
    Competing interests
    The authors declare that no competing interests exist.

Funding

No external funding was received for this work.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Wolf-Dietrich Heyer, University of California, Davis, United States

Version history

  1. Preprint posted: November 22, 2020 (view preprint)
  2. Received: April 19, 2021
  3. Accepted: July 7, 2021
  4. Accepted Manuscript published: July 8, 2021 (version 1)
  5. Version of Record published: July 19, 2021 (version 2)

Copyright

© 2021, Fuchs et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Robert P Fuchs
  2. Asako Isogawa
  3. Joao A Paulo
  4. Kazumitsu Onizuka
  5. Tatsuro Takahashi
  6. Ravindra Amunugama
  7. Julien P Duxin
  8. Shingo Fujii
(2021)
Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide
eLife 10:e69544.
https://doi.org/10.7554/eLife.69544

Share this article

https://doi.org/10.7554/eLife.69544

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