Mode of action of the <i>Spiroplasma</i> CpG methylase M.SssI

Renbaum, Paul; Razin, Aharon

doi:10.1016/0014-5793(92)81201-v

Cited by 44 publications

(37 citation statements)

References 17 publications

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“…Previous studies of M.SssI-methylated DNA with methylation-sensitive restriction endonucleases revealed that multisite substrate DNA is converted to fully endonuclease-resistant products with very little intermediate observed during the course of the reaction (26,27). As expected, our bisulfite analysis directly demonstrated that M.SssI methylates the unmethylated substrate DNA (UM) in an all-or-nothing manner.…”

Section: Enzymatic Activities Of Recombinant Dnmt1-fl and Dnmt1-dn-supporting

confidence: 58%

Processive Methylation of Hemimethylated CpG Sites by Mouse Dnmt1 DNA Methyltransferase

Vilkaitis

Suetake

Klimašauskas

et al. 2005

Journal of Biological Chemistry

185

164

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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...

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Section: Enzymatic Activities Of Recombinant Dnmt1-fl and Dnmt1-dn-supporting

confidence: 58%

Processive Methylation of Hemimethylated CpG Sites by Mouse Dnmt1 DNA Methyltransferase

Vilkaitis

Suetake

Klimašauskas

et al. 2005

Journal of Biological Chemistry

185

164

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“…The MHhaI and M.EcoRI both show significant conformational changes upon binding DNA (9,17 (21). Since neither enzyme shows processive catalysis (22,23), these enzymes must dissociate from the DNA as the enzyme-S-adenosylhomocysteine complex subsequent to catalysis.…”

mentioning

confidence: 99%

“…The M.HhaI consists of a 327-aa polypeptide folded into two domains; the DNA is bound into the intervening cleft with the major groove contacted exclusively through one protein domain, while the larger catalytic domain contains the cofactor binding site and faces the minor groove (11). The M.HhaI contains 10 conserved amino acid regions that appear in all cytosine C5 DNA methyltransferases (15 (22,23), these enzymes must dissociate from the DNA as the enzyme-S-adenosylhomocysteine complex subsequent to catalysis.We used scanning force microscopy (SFM) and gel shift analysis to determine the extent to which both enzymes bend DNA. These complementary methods show that only M.EcoRI bends DNA, and this distortion of DNA conformation is important for sequence-specific DNA recognition.…”

mentioning

confidence: 99%

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

Garcia

Bustamante²,

Reich³

1996

Proc. Natl. Acad. Sci. U.S.A.

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DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C5 DNA methyltransferase, causes only a 20 bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52°. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.Protein-mediated distortions of canonical B-DNA can contribute to DNA recognition, as demonstrated for the bacteriophage A integration host factor (1), catabolite activator protein (2-4), and the TATA binding protein (5-7). For sequence-specific enzymes, such distortions may further contribute to the correct assembly of active site residues and provide access to specific DNA moieties. A comparison of DNA substrates derived from enzyme-DNA cocrystal structures ( Fig. 1) reveals diverse enzyme-induced DNA conformations. An unprecedented example of enzyme-mediated alteration in DNA conformation was recently described for the HhaI cytosine C5 DNA methyltransferase (M.HhaI), which modifies the DNA sequence GCGC at the first cytosine (11). The cocrystal structure of the covalent enzyme-DNA complex, obtained by incorporating a mechanism-based inhibitor in place of the target cytosine, clearly shows the cytosine analog to be extrahelical while the remaining DNA is in the B-DNA conformation (Fig. 1D). The resulting orientation of the target cytosine presumably facilitates access for subsequent chemical modification by the enzyme (11). The MHhaI provides an alternative paradigm to the large-scale DNA conformational changes observed in other protein-DNA complexes; similar mechanisms have recently been suggested for exonuclease III (12), uracil-DNA glycosylase (13), and DNA photolyase (14). How other classes of methyltransferases modify their DNA is unknown.We examined the M.HhaI and an adenine N6 methyltransferase (MEcoRI, GAATTC, modifies the underlined adenine) to determine how different classes of DNA methyltransferases affect DNA structure, and in particular, the extent to which these enzymes bend DNA. The M.HhaI consists of a 327-aa polypeptide folded into two domains; the DNA is bound into the intervening cleft with the major groove contacted exclusively through one protein domain, while the larger catalytic domain contains the cofactor binding site and faces the minor groove (11). The M.HhaI contains 10 conserved amino acid regions that appear in all cytosine C5 DNA methyltransferases (15 (22,23), these enzymes must dissociate from the DNA as the enzyme-S-adenosylhomocysteine complex subsequent to catalysis.We used scanning force microscopy (SFM) and gel shift analysis to determine the extent to which both enzymes bend DNA. These complementary met...

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“…It has been reported that this enzyme methylates DNA in a processive manner, resembling mammalian methylases rather than other bacterial methylases [27].…”

Section: Resultsmentioning

confidence: 99%

Selective inhibition of cytosine‐DNA methylases by polyamines

1995

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We have advanced the hypothesis that polyamines affect DNA methylation and thus promote the expression of developmentally controlled genes. We demonstrate that the activity of cytosine-DNA methyltransferases HpaII, HhaI, HaeIII and SssI is inhibited by physiological concentrations of polyamines.On the other hand, activity of the adenine-DNA methyltransferase EcoRI, and restriction enzymes HpaII, HhaI, HaeIII and EcoRI, is insensitive to polyamine concentrations up to 40 mM.Our results indicate that the effect of polyamines on cytosine-DNA methyltranferases is rather selective and suggest a possible mode of action in vivo.

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Mode of action of the Spiroplasma CpG methylase M.SssI

Cited by 44 publications

References 17 publications

Processive Methylation of Hemimethylated CpG Sites by Mouse Dnmt1 DNA Methyltransferase

Processive Methylation of Hemimethylated CpG Sites by Mouse Dnmt1 DNA Methyltransferase

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

Selective inhibition of cytosine‐DNA methylases by polyamines

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