Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a

Tomoki Yokochi, Keith D Robertson

Research output: Contribution to journalArticle

117 Citations (Scopus)

Abstract

DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.

Original languageEnglish (US)
Pages (from-to)11735-11745
Number of pages11
JournalJournal of Biological Chemistry
Volume277
Issue number14
DOIs
StatePublished - Apr 5 2002
Externally publishedYes

Fingerprint

Methylation
Methyltransferases
DNA Methylation
DNA
Substrates
S-Adenosylmethionine
Baculoviridae
Substrate Specificity
Epigenomics
Assays
Maintenance
Kinetics
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a. / Yokochi, Tomoki; Robertson, Keith D.

In: Journal of Biological Chemistry, Vol. 277, No. 14, 05.04.2002, p. 11735-11745.

Research output: Contribution to journalArticle

@article{7167340f92ff4073b7c776f14b743c05,
title = "Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a",
abstract = "DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.",
author = "Tomoki Yokochi and Robertson, {Keith D}",
year = "2002",
month = "4",
day = "5",
doi = "10.1074/jbc.M106590200",
language = "English (US)",
volume = "277",
pages = "11735--11745",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "14",

}

TY - JOUR

T1 - Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a

AU - Yokochi, Tomoki

AU - Robertson, Keith D

PY - 2002/4/5

Y1 - 2002/4/5

N2 - DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.

AB - DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-L-methionine.

UR - http://www.scopus.com/inward/record.url?scp=0037023761&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0037023761&partnerID=8YFLogxK

U2 - 10.1074/jbc.M106590200

DO - 10.1074/jbc.M106590200

M3 - Article

C2 - 11821381

AN - SCOPUS:0037023761

VL - 277

SP - 11735

EP - 11745

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 14

ER -