Crosstalk between repair pathways elicits Double Strand Breaks in alkylated DNA and implications for the action of temozolomide
- INSERM / AMU, France
- CRCM Marseille, France
- Harvard Medical School, United States
- Tohoku Univ, Sendai, Japan
- Kyushu Univ, Fukuoka, Japan
- Harvard, BCMP, United States
- 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
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
-
- 1,755
- views
-
- 195
- downloads
-
- 18
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Citations by DOI
-
- 18
- citations for umbrella DOI https://doi.org/10.7554/eLife.69544
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)
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
