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Molecular and Cellular Biology, October 2006, p. 7575-7586, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01887-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DNA Methyltransferase 1 Knockdown Activates a Replication Stress Checkpoint

Alexander Unterberger,1 Stephen D. Andrews,1 Ian C. G. Weaver,2,3 and Moshe Szyf1,3*

Department of Pharmacology and Therapeutics,1 McGill Program for the Study of Behavior, Genes and Environment, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec H3G 1Y6, Canada,3 Douglas Hospital Research Center, 6875 LaSalle Blvd, Montréal, Québec H4H 1R3, Canada2

Received 26 September 2005/ Returned for modification 2 November 2005/ Accepted 21 July 2006

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), {gamma}H2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.

* Corresponding author. Mailing address: Room 1309, Department of Pharmacology and Therapeutics, McGill University, 3655 Sir William Osler Promenade, Montréal, Québec, Canada H3G 1Y6. Phone: (514) 398-7107. Fax: (514) 398-6690. E-mail: moshe.szyf{at}mcgill.ca.

Molecular and Cellular Biology, October 2006, p. 7575-7586, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01887-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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  • Palii, S. S., Van Emburgh, B. O., Sankpal, U. T., Brown, K. D., Robertson, K. D. (2008). DNA Methylation Inhibitor 5-Aza-2'-Deoxycytidine Induces Reversible Genome-Wide DNA Damage That Is Distinctly Influenced by DNA Methyltransferases 1 and 3B. Mol. Cell. Biol. 28: 752-771 [Abstract] [Full Text]  
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