DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of fulllength Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that fulllength Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to fulllength Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.In mammals, position 5 of cytosine residues in CpG sequences in genomic DNA is usually methylated (1). DNA methylation is one of the major epigenetic modifications that plays crucial roles in embryonic development, cell differentiation, and genomic imprinting through regulation of chromatin modification resulting in gene silencing (2). Aberrant methylation leads to human diseases, ICF (immunodeficiency centromeric region instability and facial anomalies) syndrome (3-5), and development of cancers (6). In vertebrates, two types of DNA methyltransferase activities have been reported; de novo and maintenance types. In mouse, de novo-type DNA methylation activity creates gene-specific methylation patterns at the implantation stage of embryogenesis (4), and maintenance-type activity ensures clonal transmission of lineage-specific methylation patterns during replication. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of methylation patterns at an early stage of embryogenesis, while Dnmt1 is responsible for the maintenance of methylation patterns once formed (7,8).Dnmt1 favors methylating the hemimethylated state of CpG sites (9), which appears just after the replication and repair steps. It is reported that Dnmt1 exists around replication foci (10, 11), and binds to proliferating cell nuclear antigen (PCNA) 1 (12), a prerequisite factor for replication and repair, with the sequence motif at 160 -172. Interestingly, PCNA facilitates the hemimethylation activity of Dnmt1 (13). It is also reported that the N-terminal 1-343 sequence binds to DNA (14), and the amino acid residues 284 -287 of human DNMT...

DNA cytosine-5-methyltransferase HhaI recognizes the GCGC sequence and flips the inner cytosine out of DNA helix and into the catalytic site for methylation. The 5-phosphate of the flipped out cytosine is in contact with the conserved Thr-250 from the target recognition domain. We have produced 12 mutants of Thr-250 and examined their methylation potential in vivo. Six active mutants were subjected to detailed biochemical and structural studies. Mutants with similar or smaller side chains (Ser, Cys, and Gly) are very similar to wild-type enzyme in terms of steady-state kinetic parameters k cat , K m DNA , K m AdoMet. In contrast, the mutants with bulkier side chains (Asn, Asp, and His) show increased K m values for both substrates. Fluorescence titrations and stoppedflow kinetic analysis of interactions with duplex oligonucleotides containing 2-aminopurine at the target base position indicate that the T250G mutation leads to a more polar but less solvent-accessible position of the flipped out target base. The x-ray structure of the ternary M.HhaI(T250G)⅐DNA⅐AdoHcy complex shows that the target cytosine is locked in the catalytic center of enzyme. The space created by the mutation is filled by water molecules and the adjacent DNA backbone atoms dislocate slightly toward the missing side chain. In aggregate, our results suggest that the side chain of Thr-250 is involved in constraining the conformation the DNA backbone and the target base during its rotation into the catalytic site of enzyme.

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-L-methionine (AdoMet) and product S-adenosyl-L-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. ), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k chem . We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k chem ) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. Cytosine methylation in DNA is catalyzed by DNA methyltransferases (MTases) 1 that transfer methyl groups from the ubiquitous donor S-adenosyl-L-methionine (AdoMet) producing modified cytosines with a methyl group at either C-5 or N-4 (1). In higher organisms, where only 5-methylcytosine is found, DNA methylation is essential for controlling a number of cellular processes including transcription, genomic imprinting, developmental regulation, mutagenesis, DNA repair, and chromatin organization (2). Aberrations in cytosine-5 methylation correlate with human genetic disease, and therefore, the MTases are potent candidate targets for developing new therapies (3). In prokaryotes, MTases are usually but not exclusively found as components of restriction modification systems (1).Besides their important physiological role, the MTases are attractive models for the study of protein-DNA interactions, a central event in many biological processes. The major advantages of bacterial C5-MTases as model systems are as follows: (a) wide diversity of targets recognized (over 200 specificities known); (b) ability to promote covalent reactions within the DNA; (c) their relatively simple molecular organization; and (d) high level of sequence and structural homology with eukaryotic enzymes. It is not surprising that most evidence of the catalytic mechanism of cytosine-5 methylation has been obtained from the studies of prokaryotic MTases. A particular example is HhaI MTase, a component of a type II restriction-modification system from Haemophilus haemolyticus. M.HhaI recognizes the tetranucleotide sequence GCGC and methylates the inner cytosine residue (boldface) and is one of the smallest in the C5-MTase family. This enzyme has been extensively examined by employing a variety of methods. Interaction with the substrates was shown to lead to dramatic conformational changes in both the bound DNA and the enzyme itself. MTase-mediated rotation of the target nucleotide out of the DNA helix (baseflipping) serves to deliver the base into a concave catalytic site in the enzyme (4). Subsequent massive movement of t...

Access to a nucleotide by its rotation out of the DNA helix (base flipping) is used by numerous DNA modification and repair enzymes. Despite extensive studies of the paradigm HhaI methyltransferase, initial events leading to base flipping remained elusive. Here we demonstrate that the replacement of the target C:G pair with the 2-aminopurine:T pair in the DNA or shortening of the side chain of Gln237 in the protein severely perturb base flipping, but retain specific DNA binding. Kinetic analyses and molecular modeling suggest that a steric interaction between the protruding side chain of Gln237 and the target cytosine in B-DNA reduces the energy barrier for flipping by 3 kcal/mol. Subsequent stabilization of an open state by further 4 kcal/mol is achieved through specific hydrogen bonding of the side chain to the orphan guanine. Gln237 thus plays a key role in actively opening the target C:G pair by a "push-and-bind" mechanism.