The Inherent Processivity of the Human de Novo Methyltransferase 3A (DNMT3A) Is Enhanced by DNMT3L

Celeste Holz-Schietinger,Norbert O Reich

doi:10.1074/jbc.m110.142513

Celeste Holz-Schietinger, Norbert O Reich

Open Access

https://doi.org/10.1074/jbc.m110.142513

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Abstract

Human DNMT3A is responsible for de novo DNA cytosine methylation patterning during development. Here we show that DNMT3A methylates 5-8 CpG sites on human promoters before 50% of the initially bound enzyme dissociates from the DNA. Processive methylation is enhanced 3-fold in the presence of DNMT3L, an inactive homolog of DNMT3A, therefore providing a mechanism for the previously described DNMT3L activation of DNMT3A. DNMT3A processivity on human promoters is also regulated by DNA topology, where a 2-fold decrease in processivity was observed on supercoiled DNA in comparison with linear DNA. These results are the first observation that DNMT3A utilizes this mechanism of increasing catalytic efficiency. Processive de novo DNA methylation provides a mechanism that ensures that multiple CpG sites undergo methylation for transcriptional regulation and silencing of newly integrated viral DNA.

Highlights

Committee (University of California). □S The on-line version of this article contains supplemental Figs. 1– 4 and Table 1. 1 To whom correspondence should be addressed
We investigated the enzymatic properties of the human de novo methyltransferase DNMT3A and determined its processivity using pulse-chase assays and kinetic modeling and determining kcat values for substrates with a varying number of CpG sites
We recently showed that the N-terminal PWWP domain in DNMT3A binds DNA, which may play a role in the processive mechanism of the enzyme [33]

Summary

Introduction

Committee (University of California). □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1– 4 and Table 1. 1 To whom correspondence should be addressed. Many mechanisms that control de novo methyltransferase specificity have been proposed, including cis-acting signals in the DNA sequence [12], DNA topology [13], histone modifications [14], interactions between DNMTs [15], auxiliary proteins [16], or RNA molecules [17]. The methylation of multiple, proximal CpG sites during a single binding event (processive catalysis) by DNMT3A or DNMT3B may contribute to the localized acquisition of methylation marks. We investigated the enzymatic properties of the human de novo methyltransferase DNMT3A and determined its processivity using pulse-chase assays and kinetic modeling and determining kcat values for substrates with a varying number of CpG sites. We found that DNMT3A is processive, spreading methylation to distal CpG sites. The data presented support a model of DNA methylation spreading through the action of processive catalysis

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