Crosstalk between repair pathways elicits double-strand breaks in alkylated dna and implications for the action of temozolomide

Robert P. Fuchs, Asako Isogawa, Joao A. Paulo, Kazumitsu Onizuka, Tatsuro Takahashi, Ravindra Amunugama, Julien P. Duxin, Shingo Fujii

Research output: Contribution to journalArticlepeer-review

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 (O6 mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O6 mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O6 mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6 mG: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 increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6 mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.

Original languageEnglish
Article numbere69544
JournaleLife
Volume10
DOIs
Publication statusPublished - 2021 Jul

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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