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.

摘要: 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 fibre assay to study impact on fork progression. We found that alkylator methyl methanesulfonate (MMS) inhibits elongation a manner is dose dependent and related overall alkylation grade. Replication seem be completely blocked as no nucleotide incorporation can detected following 1 h MMS treatment. A high 5 mM caffeine, inhibiting most damage signalling, decreases rates but does not reverse MMS-induced inhibition, showing block independent signalling. Furthermore, progression correlate with level single-strand breaks. Overexpression O(6)-methylguanine (O6meG)-DNA methyltransferase protein, responsible removing toxic alkylation, O6meG, did affect exposure N-methyl-N'-nitro-N-nitrosoguanidine. This demonstrates O6meG lesions are efficiently bypassed mammalian cells. In addition, find gammaH2AX foci co-localise 53BP1 newly replicated areas, suggesting double-strand breaks formed at MMS-blocked forks. Altogether, our data suggest N-alkylations during physically cells, causing formation replication-associated lesions, likely