The Dnmt1 Family

DNA methyltransferase is a comparatively large molecule of approximately 190 kDa containing 1620 amino acids.40 Enzymatically catalyzed DNA methy-lation is a covalent modification of DNA in which a methyl group is transferred from S-adenosylmethionine to the C-5 position of cytosine. Early studies41 with a prokaryotic methyltransferase (HhaI) showed that the methyl transfer reaction proceeds by an ordered Bi-Bi kinetic mechanism involving the transient formation of a covalent adduct between the enzyme and the methyl donor, S-adenosylmethionine. After transfer of the methyl group to cytosine of DNA the demethylated donor molecule dissociates followed by release of methylated DNA. All DNA methyltransferases that have been studied appear to follow a similar reaction mechanism.

More recently, expression, purification, and characterization of full length recombinant human DNMT142 showed that the enzyme prefers hemimethylated DNA compared to unmethylated DNA as a substrate. Under optimal conditions, the preference for methylation between methylated DNA averaged about 15-fold greater than that of unmethylated DNA. DNMT1 was capable of both de novo and maintenance methylation at CG sites, and could also maintain methylation of some non-CG sites.

The carboxyl-terminus of the enzyme represents its catalytic domain, while the amino-terminus has several functions including a targeting sequence that directs it to replication foci40 (Figure 6.4). Targeting of the DNA methyltransferase to replication foci is believed to permit copying of methylation patterns from parent to newly synthesized DNA of offspring. Functionally, Dnmtl was found to exhibit a 5- to 30-fold preference for hemimethylated DNA over completely un-methylated DNA. To determine whether de novo methylation and maintenance methylation were performed by the same or different enzymes, a null mutation of this enzyme was generated in mice.43,44 The null mutant was viable, but retained some capacity (approximately one-third of the wild type) for de novo methyltransferase activity, suggesting the presence of one or more other DNA

'Regulatory' domain

Catalytic domain

PBD Replication foci CXXCXXC domain

'Regulatory' domain Catalytic domain

PWWP ATRX

1620 amino acids

391 amino acids

912 amino acids

865 amino acids

'Regulatory' domain

Catalytic domain

PBD Replication foci CXXCXXC domain

'Regulatory' domain Catalytic domain

PWWP ATRX

PWWP ATRX

Figure 6.4 Human DNA methyltransferase proteins (DNMTs). All known DNMTs share a highly conserved C-terminal catalytic domain. The regulatory domain located at the N-terminus of DNMT1 differs from the domains of DNMT3A and DNMT3B. Human DNMT3A and DNMT3B are highly homologous and are probably products of gene duplication. DNMT1 shows a strong preference for hemi-methylated DNA (maintenance methylation) while DNMT3A and DNMT3B show equal activity for unmethylated and hemimethylated DNA (de novo methylation). (Sources: Adapted from Bestor,40 Okano et al., 1 Bird and Wolffe,61 and Brueckner and Lyko.73)

PWWP ATRX

Figure 6.4 Human DNA methyltransferase proteins (DNMTs). All known DNMTs share a highly conserved C-terminal catalytic domain. The regulatory domain located at the N-terminus of DNMT1 differs from the domains of DNMT3A and DNMT3B. Human DNMT3A and DNMT3B are highly homologous and are probably products of gene duplication. DNMT1 shows a strong preference for hemi-methylated DNA (maintenance methylation) while DNMT3A and DNMT3B show equal activity for unmethylated and hemimethylated DNA (de novo methylation). (Sources: Adapted from Bestor,40 Okano et al., 1 Bird and Wolffe,61 and Brueckner and Lyko.73)

methyltransferase(s) in Dnmtl knockout cells.44 The Dnmtl knockout mutation was also found to cause early embryonic lethality indicating that DNA methylation was crucial for normal mammalian development.43'45 In addition, disruption of Dnmtl resulted in abnormal imprinting, and derepression of endogenous retroviruses (summarized in Robertson and Jones46). Other targeted mutations in Dnmtl produced a number of additional unique phenotypes.40

The human homolog, DNMT1, mapped to chromosome 19p13.2-13.3.47

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