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

• Published in: eLife 2021-07, Vol.10
• Main Authors: Fuchs, Robert P, Isogawa, Asako, Paulo, Joao A, Onizuka, Kazumitsu, Takahashi, Tatsuro, Amunugama, Ravindra, Duxin, Julien P, Fujii, Shingo
• Format: Article
• Language: English
• Subjects: Adducts; alkylating agents; Alkylation; Analysis; Animals; Antimitotic agents; Antineoplastic agents; Base excision repair; base excision repair crosstalk; binding proteins discovery; Biochemistry and Chemical Biology; Cell cycle; Chromosomes and Gene Expression; Cytotoxicity; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA biosynthesis; DNA Breaks, Double-Stranded; DNA damage; DNA Mismatch Repair; DNA Repair; DNA Replication; Gene Expression; Glioblastoma; Humans; Life Sciences; Methylguanine; Mismatch repair; N-Methyl-N-nitrosourea; N3-Methyladenine; Plasmids; Proteins; Replication; sn1 versus sn2; Temozolomide; Temozolomide - metabolism; Temozolomide - pharmacology; Thymine; unbiased dna; Xenopus
• ISSN: 2050-084X; 2050-084X
• DOI: 10.7554/eLife.69544

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 O -methylguanine (O mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in 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 O 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 O 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.