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Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface
journal contribution
posted on 2016-10-21, 00:00 authored by Allison
B. Norvil, Christopher J. Petell, Lama Alabdi, Lanchen Wu, Sandra Rossie, Humaira GowherThe catalytic domains
of the de novo DNA methyltransferases
Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique
biochemical properties could potentially contribute to differences
in the substrate preferences or biological functions of these enzymes.
Dnmt3a-C forms tetramers through interactions at the dimer interface,
which also promote multimerization on DNA and cooperativity. Similar
to the case for processive enzymes, cooperativity allows Dnmt3a-C
to methylate multiple sites on the same DNA molecule; however, it
is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive
mechanism. The importance of the tetramer structure and cooperative
mechanism is emphasized by the observation that the R882H mutation
in the dimer interface of DNMT3A is highly prevalent in acute myeloid
leukemia and leads to a substantial loss of its activity. Under conditions
that distinguish between cooperativity and processivity, we show that
in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative
and confirm the processivity of Dnmt3b-C and the full length Dnmt3b
enzyme. Whereas the R878H mutation (mouse homologue of R882H) led
to the loss of cooperativity of Dnmt3a-C, the activity and processivity
of the analogous Dnmt3b-C R829H variant were comparable to those of
the wild-type enzyme. Additionally, buffer acidification that attenuates
the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C
activity. Taken together, these results demonstrate an important mechanistic
difference between Dnmt3b and Dnmt3a and suggest that interactions
at the dimer interface may play a limited role in regulating Dnmt3b-C
activity. These new insights have potential implications for the distinct
biological roles of Dnmt3a and Dnmt3b.