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

eLife

Asako Isogawa¹  Shingo Fujii¹  Julien P Duxin²  Ravindra Amunugama²  Robert P Fuchs²  Joao A Paulo³  Tatsuro Takahashi⁴  Kazumitsu Onizuka⁵ 

[1] Cancer Research Center of Marseille, UMR7258, CNRS, Marseille, France;Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, United States;Department of Cell Biology, Harvard Medical School, Boston, United States;Faculty of Science, Kyushu University, Fukuoka, Japan;Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan;

关键词: mismatch repair;    base excision repair crosstalk;    sn1 versus sn2;    alkylating agents;    unbiased dna;    binding proteins discovery;    protein-dna;    pull-down assay;    closely-spaced dna lesions;    Xenopus;    Other;   

DOI  :  10.7554/eLife.69544

来源: eLife Sciences Publications, Ltd

【 摘 要 】

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) adducts. 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 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 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 increases 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 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.

【 授权许可】

CC BY