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.

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.

Metrics

  • 1,696
    views
  • 183
    downloads
  • 17
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Mai Nguyen, Elda Bauda ... Cecile Morlot
    Research Article

    Teichoic acids (TA) are linear phospho-saccharidic polymers and important constituents of the cell envelope of Gram-positive bacteria, either bound to the peptidoglycan as wall teichoic acids (WTA) or to the membrane as lipoteichoic acids (LTA). The composition of TA varies greatly but the presence of both WTA and LTA is highly conserved, hinting at an underlying fundamental function that is distinct from their specific roles in diverse organisms. We report the observation of a periplasmic space in Streptococcus pneumoniae by cryo-electron microscopy of vitreous sections. The thickness and appearance of this region change upon deletion of genes involved in the attachment of TA, supporting their role in the maintenance of a periplasmic space in Gram-positive bacteria as a possible universal function. Consequences of these mutations were further examined by super-resolved microscopy, following metabolic labeling and fluorophore coupling by click chemistry. This novel labeling method also enabled in-gel analysis of cell fractions. With this approach, we were able to titrate the actual amount of TA per cell and to determine the ratio of WTA to LTA. In addition, we followed the change of TA length during growth phases, and discovered that a mutant devoid of LTA accumulates the membrane-bound polymerized TA precursor.

    1. Biochemistry and Chemical Biology
    2. Computational and Systems Biology
    Shinichi Kawaguchi, Xin Xu ... Toshie Kai
    Research Article

    Protein–protein interactions are fundamental to understanding the molecular functions and regulation of proteins. Despite the availability of extensive databases, many interactions remain uncharacterized due to the labor-intensive nature of experimental validation. In this study, we utilized the AlphaFold2 program to predict interactions among proteins localized in the nuage, a germline-specific non-membrane organelle essential for piRNA biogenesis in Drosophila. We screened 20 nuage proteins for 1:1 interactions and predicted dimer structures. Among these, five represented novel interaction candidates. Three pairs, including Spn-E_Squ, were verified by co-immunoprecipitation. Disruption of the salt bridges at the Spn-E_Squ interface confirmed their functional importance, underscoring the predictive model’s accuracy. We extended our analysis to include interactions between three representative nuage components—Vas, Squ, and Tej—and approximately 430 oogenesis-related proteins. Co-immunoprecipitation verified interactions for three pairs: Mei-W68_Squ, CSN3_Squ, and Pka-C1_Tej. Furthermore, we screened the majority of Drosophila proteins (~12,000) for potential interaction with the Piwi protein, a central player in the piRNA pathway, identifying 164 pairs as potential binding partners. This in silico approach not only efficiently identifies potential interaction partners but also significantly bridges the gap by facilitating the integration of bioinformatics and experimental biology.