Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage |
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Authors: | Petra Groth Muntasir Mamun Majumder Fredrik Johansson Thomas Helleday |
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Institution: | 1 Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden2 Gray Institute for Radiation Oncology and Biology, University of Oxford, Old Road Campus Research Building, Off Roosevelt Drive, Oxford OX3 7DQ, UK |
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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 O6-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 γH2AX 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. |
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Keywords: | MMS methyl methanesulfonate O6meG O6-methylguanine MNNG N-methyl-N&prime -nitro-N-nitrosoguanidine N7meG N7-methylguanine N3meA N3-methyladenine BER base excision repair SSB single-strand break MGMT O6meG-DNA methyltransferase DSB double-strand break HR homologous recombination TLS translesion synthesis CldU 5-chlorodeoxyuridine IdU 5-iododeoxyuridine GFP green fluorescent protein FACS fluorescence-activated cell sorting PFGE pulse-field gel electrophoresis HBSS Hanks balanced salt solution PBS phosphate-buffered saline PBS-T PBS containing 0 1% Triton X-100 |
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