Murine DNA (Cytosine-5-)-methyltransferase:  Steady-State and Substrate Trapping Analyses of the Kinetic Mechanism

Abstract

DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in KmDNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Double-reciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dI.dC-dI.dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M. HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

“…Consistent with our result, all cytosine methyltransferases studied thus far (e.g. M.HhaI, M.MspI, murine Dnmt1, and human DNMT1) follow an Ordered Bi Bi mechanism with DNA as the leading substrate (14,30,33,34). In contrast, bacterial adenine methyltransferases as a group may exhibit multiple distinct mechanisms (46,59,60).…”

“…Mammalian maintenance DNA methyltransferase DNMT1 has been extensively studied biochemically and enzymatically (14,(27)(28)(29)(30)(31). DNMT1 prefers hemimethylated dsDNA and its kinetic constants have been reported (14,28,29).…”

Characterization of the Human DNA Methyltransferase Splice Variant Dnmt1b

“…Both the DNA MeTase isoforms, Dnmt1 and Dnmt1b, have similar Michaelis constants for both hemimethylated (K m mCG ) and nonmethylated DNA (K m CG ) as well as Michaelis constants for the cofactor S-adenosyl-L-methionine (K m SAM ) (Table I). Moreover, these values are comparable to those determined for the murine Dnmt1 protein (29,36). RNase protection analysis showed that Dnmt1b mRNA is expressed at a lower level than Dnmt1 in human cells (Fig.…”

Bacteriophage T4 Dam DNA-[N6-adenine]Methyltransferase

“…MTases (10). However, a random mechanism has been proposed for the human Dnmt1 MTase (9) and HhaI (7).…”