The inherent processivity of the human de novo methyltransferase 3A (DNMT3A) is enhanced by DNMT3L - PubMed (original) (raw)

The inherent processivity of the human de novo methyltransferase 3A (DNMT3A) is enhanced by DNMT3L

Celeste Holz-Schietinger et al. J Biol Chem. 2010.

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.

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Figures

FIGURE 1.

FIGURE 1.

DNMT3A is processive on a variety of substrates, as shown using a pulse-chase assay. A, poly(dI-dC) (5 μ

m

bp). B, EFS1 human promoter (10 μ

m

bp). C, bacterial supercoiled plasmid pBR322 (10 μ

m

bp). D and E, the p21 human promoter in the pCpGL plasmid, linearized (20 μ

m

bp) (D) and supercoiled (20 μ

m

bp) (E). Substrate was added at time 0 to start the reaction. Full-length DNMT3A is at 50 n

m

, and AdoMet is at 2 μ

m

. ■ = only substrate; ● = substrate and then 200 μ

m

bp pCpGL at 20 min; ▴ = substrate and 200 μ

m

bp pCpGL at the start of the reaction.

FIGURE 2.

FIGURE 2.

The number of CpGs in a promoter increases the rate of product formation. The turnover rate was calculated for DNMT3A on substrates with a varying CpG sites. A, _k_cat values were compared with the number of CpG sites in the DNA substrate. Each point is the _k_cat value for the human promoters and synthesized substrate with one CpG site. The inset includes poly(dI-dC). The regression line shows _R_2 = 0.85, with _R_2 = 0.98 for the inset. B, comparing the length of DNA with _k_cat values shows little correlation (_R_2 = 0.45). C–E, substrate inhibition plots for the PCR-amplified human promoters, which are representative plots for obtaining _V_max data. _V_max values were divided by the amount of active enzyme in the reaction to obtain _k_cat values.

FIGURE 3.

FIGURE 3.

Commitment for processivity. A, processivity assays were performed by adding chaser DNA (pCpGL) at different times into the reaction. Reactions were run with full-length DNMT3A at 50 n

m

, AdoMet at 2 μ

m

, substrate DNA (poly(dI-dC)) at 5 μ

m

, and chaser DNA (pCpGL) at 200 μ

m

. The timing of adding chaser DNA varied in each reaction, as indicated by arrows. Enzyme was preincubated with AdoMet for 3 min before DNA addition (similar results were seen by preincubating with DNA, data not shown). ■ = poly(dI-dC) at 5 μ

m

; ● = poly(dI-dC) at 5 μ

m

with 200 μ

m

pCpGL at 20 min; ♦ = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at 15 min, □ = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at 10 min; ◇ = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at 5 min, ▴ = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at start of reaction. B, schematic of the actions of an enzyme when chaser DNA is added at different times.

FIGURE 4.

FIGURE 4.

The N terminus of DNMT3A is not needed for processive catalysis. A, the pulse-chase processivity assay was preformed on the catalytic domain of DNMT3A. Chaser DNA (pCpGL) was added 20 min into the reaction. Enzyme was at 50 n

m

, AdoMet was at 2 μ

m

, substrate DNA (poly(dI-dC)) was at 5 μ

m

, and chaser DNA was at 200 μ

m

. ■ = poly(dI-dC) at 5 μ

m

, ● = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at 25 min, ▴ = poly(dI-dC) at 5 μ

m

and 200 μ

m

pCpGL at the start of reaction. B, schematic of the domains of DNMT3A, DNMT3A catalytic domain, and DNMT3L.

FIGURE 5.

FIGURE 5.

DNMT3L is a processivity factor of DNMT3A. A, DNMT3L activates DNMT3A, where DNMT3L is varied from 12.4 to 200 n

m

, and DNMT3A is at 50 n

m

. B, comparing DNMT3L activation on DNMT3A on a multiple recognition substrate poly(dI-dC) (25 μ

m

) and a single site substrate GCbox2 (25 μ

m

) in saturating conditions. C and D, processive assay same as in Fig. 1 but including DNMT3L. DNMT3A and DNMT3L (50 n

m

each) were preincubated for 1 h in reaction buffer with AdoMet at 2 μ

m

followed by DNA addition at time 0. C, substrate, poly(dI-dC) (5 μ

m

). D, the _p21_-pCpGL plasmid, supercoiled, was used as the substrate (20 μ

m

). ■ = only substrates; ● = substrate and then 200 μ

m

pCpGL added at 20 min; ▴ = substrate and 200 μ

m

pCpGL at start of reaction. E and F, average number of catalytic events on a substrate before dissociation (_n_½ value), with and without DNMT3L; error bars are S.D. between three independent reactions.

FIGURE 6.

FIGURE 6.

Model of de novo methylation. A depiction of DNMT3A with DNMT3L carrying out either processive or non-processive catalysis is shown.

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