Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 402, Issue 1, Pages 70-82Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2010.07.010
Keywords
N-methyl-N '-nitro-N-nitrosoguanidine; methyl methanesulfonate; mammalian cells; replication fork elongation; double-strand breaks
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Funding
- Swedish Cancer Society
- Swedish Children's Cancer Foundation
- Swedish Research Council
- Swedish Pain Relief Foundation
- Medical Research Council
- MRC [G0700730] Funding Source: UKRI
- Medical Research Council [G0700730] Funding Source: researchfish
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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-M-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gamma H2AX 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. (C) 2010 Elsevier Ltd. All rights reserved.
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