title: Methylated DNA causes a physical block to replication forks independently of damage signalling, O(6)-methylguanine or DNA single-strand breaks and results in DNA damage.
abstract: Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gammaH2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks.
authors.affiliation: Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden.
authors.forename: Petra
authors.initials: P
authors.lastname: Groth
authors.forename: Simon
authors.initials: S
authors.lastname: Ausländer
authors.forename: Muntasir Mamun
authors.initials: MM
authors.lastname: Majumder
authors.forename: Niklas
authors.initials: N
authors.lastname: Schultz
authors.forename: Fredrik
authors.initials: F
authors.lastname: Johansson
authors.forename: Eva
authors.initials: E
authors.lastname: Petermann
authors.forename: Thomas
authors.initials: T
authors.lastname: Helleday
mesh_terms: D000478:Alkylation; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects*; D004249:DNA Damage; D019175:DNA Methylation; D004261:DNA Replication / Q000187:drug effects*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006151:Guanosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000378:metabolism*
chemical_list: D006151:Guanosine; C046175:6'-O-methylguanosine; D008741:Methyl Methanesulfonate; D019853:O(6)-Methylguanine-DNA Methyltransferase