The DNMT1 Target Recognition Domain Resides in the N Terminus

Abstract

DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. Vertebrate genomes are modified by methylation of ϳ60 -80% of the cytosines residing at the CG dinucleotide sequence (1). The distribution of methylated cytosines is not random, resulting in gene-and tissue-specific patterns of methylation (2). A large body of evidence supports the hypothesis that both methylation patterns and activity of DNA methyltransferases (DNMTs) 1 play critical roles in development and in controlling genome functions such as differential gene expression, chromosome imprinting, and X-chromosome inactivation (3-5). It has also been suggested that DNMT1 is a downstream effector of many oncogenic pathways and a potential target for anticancer therapy (6 -11). We have previously demonstrated that inhibition of DNMT1 leads to an inhibition of DNA replication (12). Recently, it has been shown that DNMT1 is able to form a complex with Rb, E2F, and HDAC1 and repress E2F-responsive expression (13,14). Furthermore, it has been shown that DNMT1 can establish a transcriptional repressive complex with HDAC2 and DMAP1 at replication foci (15). These data suggest that DNMT1 has multiple functions in the cell. However, because the DNMT1 target recognition domain is unknown it is not possible to determine how these multiple functions and protein-protein interactions relate to its target specificity.If DNA methylation patterns contain significant information, there must be a mechanism that ensures its proper inheritance in cell lineages. Razin and Riggs (16) have proposed that patterns of methylation are inherited, because DNMT1 is more proficient in methylating hemimethylated DNA than nonmethylated DNA. This hypothesis has been verified by a number of experiments (17, 18). Another level of specificity is the ability of DNMT1 to recognize CG sequences almost exclusively (19,20). Thus, DNMT1 exhibits both substrate and sequence specificity.The mammalian DNMT1 is a protein postulated to be composed, based on its similarity to other cytosine DNA methyltransferases, of at least three structural components (21-26). These domains are as follows: a catalytic domain at the C terminus, an N-terminal domain that is responsible for localization of the protein to the nucleus and replication foci, and another poorly characterized central domain. It is unclear as yet which segment is responsible for determining its specificity for hemimethylated CG sequences. Previous reports have shown that when the N-terminal domain is cleaved by proteolysis, the enzyme loses its ability to discriminate between hemimethylated and un...

Processive Methylation of Hemimethylated CpG Sites by Mouse Dnmt1 DNA Methyltransferase

“…Nearly identical behavior of the truncated enzyme with all the substrates, including preferred methylation of hemimethylated DNA, clearly indicates that the N-terminal region containing the binding sequences for DNA and PCNA is dispensable for the selectivity of Dnmt1 toward hemimethylated sites. Araujo et al (25) reported that the targeting sequence for hemimethylated DNA resides in the N-terminal domain near the PCNA recognition site; however, our present results show that the N-terminal domain containing the PCNA site is dispensable for the preferential methylation of Dnmt1 toward hemimethylated DNA.…”

“…The PCNA complex with replication machinery slides on DNA (29,30), and the interaction of Dnmt1 with PCNA facilitates activity toward hemimethylated DNA (13). As well, the hemimethylated DNA targeting sequence in Dnmt1 was reported to reside near the PCNA binding site (25). Interactions of the N-terminal domain of Dnmt1 with DNA and PCNA may contribute to ensuring the immediate early methylation of the hemimethylated form of newly synthesized DNA strands to maintain methylation patterns at the replication fork.…”

“…After the methylation reaction, DNA fragments were cleaved with AflIII, ligated with the hairpin linker, treated with bisulfite, PCR-amplified with primers VA2/AA4, and then sequenced. B, among 65 clones sequenced, 14, 11, and 12 contained Dnmt1 methylation on both strands (clones [1][2][3][4][5][6][7][8][9][10][11][12][13][14], on the lower strand (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), and on the upper strand (26 -37), respectively. Symbols are as in Fig.…”

Major and Essential Role for the DNA Methylation Mark in Mouse Embryogenesis and Stable Association of DNMT1 with Newly Replicated Regions

“…A number of biochemical studies suggested that conformational change and the interaction between the N-terminal region and the C-terminal catalytic domain play important roles in DNMT1 function (1,2,17,25,38). Our results suggested that, unlike full-length DNMT1, the N-terminal domain of DNMT1 associated with unmethylated chromatin, and the RFT/TS domain was responsible for this function (Fig.…”

The Epigenetics of Adult (Somatic) Stem Cells

“…DNMT1 localizes to the replication fork 42 where it associates with PCNA (the replication processivity protein), 43 and association with the hemimethylated sequence has been localized to the N-terminal domain. 44 DNMTs 3a and 3b are classified as de novo methyltransferases; they bind to both hemimethylated and unmethylated CpG sites and add methyl groups to previously unmethylated cytosines. De novo methylation occurs extensively during early development and, once the methylation patterns are established, appear to be faithfully maintained for the life of the dividing cell.…”