Distinct DNA methylation activity of Dnmt3a and Dnmt3b towards naked and nucleosomal DNA

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.     

Stimulation effect of Dnmt3L on the DNA methylation activity of Dnmt3a2

Quantification of DNA methyltransferases Dnmt3a and Dnmt3a2, and Dnmt3L in isolated male gonocytes in day 16.5 embryos confirmed that not Dnmt3a but Dnmt3a2 and Dnmt3L were the major Dnmt3s. The expression level of Dnmt3L constituted 5- to 10-fold molar excess compared to that of Dnmt3a2. The stimulation property of the DNA methylation activity of Dnmt3a2 with Dnmt3L towards substrate DNA in naked or nucleosomes was similar to that of Dnmt3a. However, the DNA methylation activity of not Dnmt3a but Dnmt3a2 was severely inhibited at the physiological salt concentration. Interestingly, the activity of Dnmt3a2 was significantly detected in the presence of Dnmt3L even at the physiological salt concentration. This indicates that Dnmt3a2 functions only in the presence of Dnmt3L in male gonocytes, and may explain why Dnmt3L is required specifically in mouse gonocytes for DNA methylation.     

Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA.

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre-existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.     

DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, possibly by interacting with Dnmt3a and/or Dnmt3b (Hata, K., Okano, M., Lei, H., and Li, E. (2002) Development 129, 1983-1993). In the present study, the effect of DNMT3L, a human homologue of Dnmt3L, on the DNA methylation activity of mouse Dnmt3a and Dnmt3b was examined in vitro. DNMT3L enhanced the DNA methylation activity of Dnmt3a and Dnmt3b about 1.5-3-fold in a dose-dependent manner but did not enhance the DNA methylation activity of Dnmt1. Although the extents of stimulation were different, a stimulatory effect on the DNA methylation activity was observed for all of the substrate DNA sequences examined, such as those of the maternally methylated SNRPN and Lit-1 imprinting genes, the paternally methylated H19 imprinting gene, the CpG island of the myoD gene, the 5 S ribosomal RNA gene, an artificial 28-bp DNA, poly(dG-dC)-poly(dG-dC), and poly(dI-dC)-poly(dI-dC). DNMT3L could not bind to DNA but could bind to Dnmt3a and Dnmt3b, indicating that the stimulatory effect of DNMT3L on the DNA methylation activity may not be due to the guiding of Dnmt3a and Dnmt3b to the targeting DNA sequence but may comprise a direct effect on their catalytic activity. The carboxyl-terminal half of DNMT3L was found to be responsible for the enhancement of the enzyme activity.     

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.     

De novo methylation of nucleosomal DNA by the mammalian Dnmt1 and Dnmt3A DNA methyltransferases

In the cell, DNA is wrapped on histone octamers, which reduces its accessibility for DNA interacting enzymes. We investigated de novo methylation of nucleosomal DNA in vitro and show that the Dnmt3a and Dnmt1 DNA methyltransferases efficiently methylate nucleosomal DNA without dissociation of the histone octamer from the DNA. In contrast, the prokaryotic SssI DNA methyltransferase and the catalytic domain of Dnmt3a are strongly inhibited by nucleosomes. We also found that full-length Dnmt1 and Dnmt3a bind to nucleosomes much stronger than their isolated catalytic domains, demonstrating that the N-terminal parts of the MTases are required for the interaction with nucleosomes. Variations of the DNA sequence or the histone tails did not significantly influence the methylation activity of Dnmt3a. The observation that mammalian methyltransferases directly modify nucleosomal DNA provides an insight into the mechanisms by which histone tail and DNA methylation patterns can influence each other because the DNA methylation pattern can be established while histones remain associated to the DNA.     

Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog

DNA methylation plays an important role in gene silencing in mammals. Two de novo methyltransferases, Dnmt3a and Dnmt3b, are required for the establishment of genomic methylation patterns in development. However, little is known about their coordinate function in the silencing of genes critical for embryonic development and how their activity is regulated. Here we show that Dnmt3a and Dnmt3b are the major components of a native complex purified from embryonic stem cells. The two enzymes directly interact and mutually stimulate each other both in vitro and in vivo. The stimulatory effect is independent of the catalytic activity of the enzyme. In differentiating embryonic carcinoma or embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoters of the Oct4 and Nanog genes. Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog during the differentiation of pluripotent cells and mouse embryonic development. These results suggest that Dnmt3a and Dnmt3b form a complex through direct contact in living cells and cooperate in the methylation of the promoters of Oct4 and Nanog during cell differentiation. The physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development.     

In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells

3-Methyladenine (3MeA) DNA glycosylases initiate base excision repair by removing 3MeA. These glycosylases also remove a broad spectrum of spontaneous and environmentally induced base lesions in vitro. Mouse cells lacking the Aag 3MeA DNA glycosylase (also known as the Mpg, APNG or ANPG DNA glycosylase) are susceptible to 3MeA-induced S phase arrest, chromosome aberrations and apoptosis, but it is not known if Aag is solely responsible for repair of 3MeA in vivo. Here we show that in Aag^–/– cells, 3MeA lesions disappear from the genome slightly faster than would be expected by spontaneous depurination alone, suggesting that there may be residual repair of 3MeA. However, repair of 3MeA is at least 10 times slower in Aag^–/– cells than in Aag^+/+ cells. Consequently, 24 h after exposure to [³H]MNU, 30% of the original 3MeA burden is intact in Aag^–/– cells, while 3MeA is undetectable in Aag^+/+ cells. Thus, Aag is the major DNA glycosylase for 3MeA repair. We also investigated the in vivo repair kinetics of another Aag substrate, 7-methylguanine. Surprisingly, 7-methylguanine is removed equally efficiently in Aag^+/+ and Aag^–/– cells, suggesting that another DNA glycosylase acts on lesions previously thought to be repaired by Aag.     

Preferential methylation of unmethylated DNA by Mammalian de novo DNA methyltransferase Dnmt3a

DNA methylation is an epigenetic modification of DNA. There are currently three catalytically active mammalian DNA methyltransferases, DNMT1, -3a, and -3b. DNMT1 has been shown to have a preference for hemimethylated DNA and has therefore been termed the maintenance methyltransferase. Although previous studies on DNMT3a and -3b revealed that they act as functional enzymes during development, there is little biochemical evidence about how new methylation patterns are established and maintained. To study this mechanism we have cloned and expressed Dnmt3a using a baculovirus expression system. The substrate specificity of Dnmt3a and molecular mechanism of its methylation reaction were then analyzed using a novel and highly reproducible assay. We report here that Dnmt3a is a true de novo methyltransferase that prefers unmethylated DNA substrates more than 3-fold to hemimethylated DNA. Furthermore, Dnmt3a binds DNA nonspecifically, regardless of the presence of CpG dinucleotides in the DNA substrate. Kinetic analysis supports an Ordered Bi Bi mechanism for Dnmt3a, where DNA binds first, followed by S-adenosyl-l-methionine.     

Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase.

An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans. Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol. Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol. The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins. These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein.     

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC₅₀ 3–9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the K_d of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin’s ability to intercalate into DNA.     

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC–MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.     

Stoichiometry and affinity for thymine DNA glycosylase binding to specific and nonspecific DNA

Deamination of 5-methylcytosine to thymine creates mutagenic G · T mispairs, contributing to cancer and genetic disease. Thymine DNA glycosylase (TDG) removes thymine from these G · T lesions, and follow-on base excision repair yields a G · C pair. A previous crystal structure revealed TDG (catalytic domain) bound to abasic DNA product in a 2:1 complex, one subunit at the abasic site and the other bound to undamaged DNA. Biochemical studies showed TDG can bind abasic DNA with 1:1 or 2:1 stoichiometry, but the dissociation constants were unknown, as was the stoichiometry and affinity for binding substrates and undamaged DNA. We showed that 2:1 binding is dispensable for G · U activity, but its role in G · T repair was unknown. Using equilibrium binding anisotropy experiments, we show that a single TDG subunit binds very tightly to G · U mispairs and abasic (G · AP) sites, and somewhat less tightly G · T mispairs. Kinetics experiments show 1:1 binding provides full G · T activity. TDG binds undamaged CpG sites with remarkable affinity, modestly weaker than G · T mispairs, and exhibits substantial affinity for nonspecific DNA. While 2:1 binding is observed for large excess TDG concentrations, our findings indicate that a single TDG subunit is fully capable of locating and processing G · U or G · T lesions.     

Human DNA glycosylase enzyme TDG repairs thymine mispaired with exocyclic etheno-DNA adducts

Lipid peroxidation directly reacts with DNA and produces various exocyclic etheno-base DNA adducts, some of which are considered to contribute to carcinogenesis. However, the system for repairing them in humans is largely unknown. We hypothesized that etheno-DNA adducts are repaired by base excision repair initiated by DNA glycosylase. To test this hypothesis, we examined the activities of the DNA glycosylase proteins OGG1, SMUG1, TDG, NEIL1, MUTYH, NTH1, MPG, and UNG2 against double-stranded oligonucleotides containing 1,N⁶-ethenoadenine (εA), 3,N⁴-ethenocytosine (εC), butanone-ethenocytosine (BεC), butanone-ethenoguanine (BεG), heptanone-ethenocytosine (HεC), or heptanone-ethenoguanine (HεG) using a DNA cleavage assay. We found that TDG is capable of removing thymine that has mispaired with εC, BεC, BεG, HεC, or HεG in vitro. We next examined the effect of TDG against etheno-DNA adducts in human cells. TDG-knockdown cells exhibited the following characteristics: (a) higher resistance to cell death caused by the induction of etheno-DNA adducts; (b) lower repair activity for εC; and (c) a modest acceleration of mutations caused by εC, compared with the rate in control cells. All these characteristics suggest that TDG exerts a repair activity against etheno-DNA adducts in human cells. These results suggest that TDG has novel repair activities toward etheno-DNA adducts.