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Targeted Mutagenesis Results in an Activation of DNA Methyltransferase 1 and Confirms an Autoinhibitory Role of its RFTS Domain

Targeted Mutagenesis Results in an Activation of DNA Methyltransferase 1 and Confirms an... The N‐terminal regulatory part of DNA methyltransferase 1 (Dnmt1) contains a replication foci targeting sequence (RFTS) domain, which is involved in the recruitment of Dnmt1 to replication forks. The RFTS domain has been observed in a crystal structure to bind to the catalytic domain of the enzyme and block its catalytic centre. Removal of the RFTS domain led to activation of Dnmt1, thus suggesting an autoinhibitory role of this domain. Here, we destabilised the interaction of the RFTS domain with the catalytic domain by site‐directed mutagenesis and purified the corresponding Dnmt1 variants. Our data show that these mutations resulted in an up to fourfold increase in Dnmt1 methylation activity in vitro. Activation of Dnmt1 was not accompanied by a change in its preference for methylation of hemimethylated CpG sites. We also show that the Dnmt1 E572R/D575R variant has a higher DNA methylation activity in human cells after transfection into HCT116 cells, which are hypomorphic for Dnmt1. Our findings strongly support the autoinhibitory role of the RFTS domain, and indicate that it contributes to the regulation of Dnmt1 activity in cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png ChemBioChem Wiley

Targeted Mutagenesis Results in an Activation of DNA Methyltransferase 1 and Confirms an Autoinhibitory Role of its RFTS Domain

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References (28)

Publisher
Wiley
Copyright
"Copyright © 2014 Wiley Subscription Services, Inc., A Wiley Company"
ISSN
1439-4227
eISSN
1439-7633
DOI
10.1002/cbic.201300740
pmid
24532244
Publisher site
See Article on Publisher Site

Abstract

The N‐terminal regulatory part of DNA methyltransferase 1 (Dnmt1) contains a replication foci targeting sequence (RFTS) domain, which is involved in the recruitment of Dnmt1 to replication forks. The RFTS domain has been observed in a crystal structure to bind to the catalytic domain of the enzyme and block its catalytic centre. Removal of the RFTS domain led to activation of Dnmt1, thus suggesting an autoinhibitory role of this domain. Here, we destabilised the interaction of the RFTS domain with the catalytic domain by site‐directed mutagenesis and purified the corresponding Dnmt1 variants. Our data show that these mutations resulted in an up to fourfold increase in Dnmt1 methylation activity in vitro. Activation of Dnmt1 was not accompanied by a change in its preference for methylation of hemimethylated CpG sites. We also show that the Dnmt1 E572R/D575R variant has a higher DNA methylation activity in human cells after transfection into HCT116 cells, which are hypomorphic for Dnmt1. Our findings strongly support the autoinhibitory role of the RFTS domain, and indicate that it contributes to the regulation of Dnmt1 activity in cells.

Journal

ChemBioChemWiley

Published: Sep 21, 2015

Keywords: ; ; ;

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