Specificity of Dnmt1 for Methylation of Hemimethylated CpG Sites Resides in Its Catalytic Domain

Bashtrykov, Pavel; Jankevicius, Gytis; Smarandache, Anita; Jurkowska, Renata Z.; Ragozin, Sergey; Jeltsch, Albert

doi:10.1016/j.chembiol.2012.03.010

Cited by 86 publications

(67 citation statements)

References 23 publications

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“…Dnmt1 concentration was measured by NanoDrop (Thermo Scientific), the quality of preparation was confirmed by SDS-PAGE (purity >95%). Adenosyl-L-methionine (3 TBq/mMole) (PerkinElmer) and buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 100 mM KCl) using the avidin plate assay as described [13,14]. Substrates for the methylation assay were prepared by annealing of complementary 20mer DNA oligonucleotides one of which contained biotin at its 5 0 end.…”

Section: Dnmt1 Expression and Purificationmentioning

confidence: 99%

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

2012

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a b s t r a c tA recently solved Dnmt1-DNA crystal structure revealed several enzyme-DNA contacts and large structural rearrangements of the DNA at the target site, including the flipping of the non-target strand base of the base pair flanking the CpG site and formation of a non-canonical base pair between the non-target strand Gua and the flanking base pair. Here, we show that the contacts of the enzyme to the target base and the Gua:5mC base pair that are observed in the structure are very important for catalytic activity. The contacts to the non-target strand Gua are not important since its exchange by Ade stimulated activity. Except target base flipping, we could not find evidence that the DNA rearrangements have a functional role.

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Section: Dnmt1 Expression and Purificationmentioning

confidence: 99%

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

2012

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“…DNA Binding of SRA Domain Mutants-DNA binding of SRA domain mutants was investigated using an electrophoretic mobility shift assay essentially as described previously (13). Radioactively labeled hemimethylated 30-mer DNA was incubated with increasing concentrations of the SRA domain wild type and mutants (0.25, 0.5, and 1 M) for 20 min at 22°C.…”

Section: Methodsmentioning

confidence: 99%

“…Protein Expression and Purification-Mouse DNMT1 wild type (NCBI Reference Sequence NP_034196.5) and the DNMT1 E406R/D407R mutant were expressed using a baculovirus expression system according to the manufacturer's instructions (Bac-to-Bac manual, Invitrogen) and as described previously (13,16). Both proteins were cloned into pFastBacHTa as N-terminal His 6 -YFP fusions.…”

Section: Methodsmentioning

confidence: 99%

“…Structures of larger DNMT1 fragments provided evidence for complex allosteric regulation of its activity and specificity. The CXXC domain has been implicated in an autoinhibitory function (11), but the role of the CXXC domain in the context of the full-length protein has not yet been clarified (13). Additionally, the crystal structure of DNMT1(291-1620) without DNA (19) showed that the replication focus targeting sequence (RFTS) domain occupied the DNA binding cleft in the catalytic domain of DNMT1, and the CXXC domain was moved aside.…”

Section: Dnmt1mentioning

confidence: 99%

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The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism

Bashtrykov

Jankevicius

Jurkowska

et al. 2014

Journal of Biological Chemistry

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119

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Background: UHRF1-mediated targeting of DNMT1 to replicated DNA is essential for DNA methylation. Results: We show here that UHRF1 stimulates the activity and specificity of DNMT1 by an allosteric mechanism. Conclusion: UHRF1 has multiple roles that support DNA methylation including targeting and regulation of the activity and specificity of DNMT1. Significance: Regulation of DNMT1 is essential for the rapid and faithful remethylation of DNA after replication.

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“…Interference of the repressive activity results in the aberrant expression of genes (Rountree et al, 2000). Furthermore, any impendence in the normal binding of DMAP1 to DNA at CpG sites may exhibit a lack of DNMT1 activity, which affects the gene expression (Bashtrykov et al, 2012). Besides, DMAP1 also form several complexes that participate in the replication process.…”

Section: Resultsmentioning

confidence: 99%

In silico docking of methyl isocyanate (MIC) and its hydrolytic product (1, 3-dimethylurea) shows significant interaction with DNA Methyltransferase 1 suggests cancer risk in Bhopal-Gas-Tragedy survivors

Khan¹,

Senthilkumar²,

Upadhyay³

et al. 2015

Asian Pacific Journal of Cancer Prevention

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DNA methyltransferase 1 (DNMT1) is a relatively large protein family responsible for maintenance of normal methylation, cell growth and survival in mammals. Toxic industrial chemical exposure associated methylation misregulation has been shown to have epigenetic influence. Such misregulation could effectively contribute to cancer development and progression. Methyl isocyanate (MIC) is a noxious industrial chemical used extensively in the production of carbamate pesticides. We here applied an in silico molecular docking approach to study the interaction of MIC with diverse domains of DNMT1, to predict cancer risk in the Bhopal population exposed to MIC during 1984. For the first time, we investigated the interaction of MIC and its hydrolytic product (1,3-dimethylurea) with DNMT1 interacting (such as DMAP1, RFTS, and CXXC) and catalytic (SAM, SAH, and Sinefungin) domains using computer simulations. The results of the present study showed a potential interaction of MIC and 1,3-dimethylurea with these domains. Obviously, strong binding of MIC with DNMT1 interrupting normal methylation will lead to epigenetic alterations in the exposed humans. We suggest therefore that the MICexposed individuals surviving after 1984 disaster have excess risk of cancer, which can be attributed to alterations in their epigenome. Our findings will help in better understanding the underlying epigenetic mechanisms in humans exposed to MIC.

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Specificity of Dnmt1 for Methylation of Hemimethylated CpG Sites Resides in Its Catalytic Domain

Cited by 86 publications

References 23 publications

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase

The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism

In silico docking of methyl isocyanate (MIC) and its hydrolytic product (1, 3-dimethylurea) shows significant interaction with DNA Methyltransferase 1 suggests cancer risk in Bhopal-Gas-Tragedy survivors

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