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D73A
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25fold improvement in affinity to S-adenosyl-L-methionine
M168A
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4fold enhancement in the ratio of turnover number/Km-value(DNA, mainly caused be change in Km)
N39A
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4fold enhancement in the ratio of turnover number/Km-value(DNA)
Q301A
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40fold decreased Km-ratio of turnover number/Km-value(DNA) and an 80fold increased Kd(DNA)-value
V282A
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4fold improvement in catalytic turnover
N304A
4fold improved activity with S-adenosyl-L-methionine analog containing a S-butynyl group, 5fold improved actitivity with S-adenosyl-L-methionine analog containing a S-pentynyl group
Q82A
4fold improved activity with S-adenosyl-L-methionine analog containing a S-butynyl group, 16fold decrease in activity with S-adenosyl-L-methionine
Q82A/N304A
no further improvement of activity compared to single mutants
Q82A/Y254S/N304A
significantly improved actitivity with S-adenosyl-L-methionine analogs containing a S-butynyl, a S-pentynyl or a S-6-(4-aminobutanamido)hex-2-yn-1-yl group
Y254S
significantly improved actitivity with S-adenosyl-L-methionine analogs containing a S-butynyl or a S-pentynyl group
Y254S/N304A
significantly improved actitivity with S-adenosyl-L-methionine analogs containing a S-butynyl, a S-pentynyl or a S-6-(4-aminobutanamido)hex-2-yn-1-yl group
C667G
zinc-finger-domain point mutation in the CXXC motif abolishes unmethylated DNA binding activity
C667G/C670G
zinc-finger-domain point mutation in the CXXC motif abolishes unmethylated DNA binding activity
C670G
zinc-finger-domain point mutation in the CXXC motif abolishes unmethylated DNA binding activity
DELAT1-501
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to accelerate the rate of DNMT1 with the hemimethylated substrate, the 501 amino acids at the N terminus are deleted. Truncated DNMT1 exhibits a 4fold increased kcat/Km as compared with the full-length enzyme and does not require allosteric activation by hemimethylated DNA
DELTA1-501
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no stimulation by methylated DNA
DELTA1-540
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no stimulation by methylated DNA
DELTA1-580
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no stimulation by methylated DNA
H286A/R287A
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allosteric activation by methylated DNA is reduced, 6.5 fold decrease in the ratio turnover number/Km-value compared to wild-type enzyme
K284A/K285A
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allosteric activation by methylated DNA is reduced, 1.7fold decrease in the ratio turnover number/Km-value compared to wild-type enzyme
A609T
naturally occuring mutation causing recessive genetic disorder, ICF syndrome, the mutation does not affect the association of Dnmt3b with Mbd4 or Tdg
S277P
naturally occuring mutation causing recessive genetic disorder, ICF syndrome, the mutation does not affect the association of Dnmt3b with Mbd4 or Tdg
V612A
naturally occuring mutation causing recessive genetic disorder, ICF syndrome, the mutation does not affect the association of Dnmt3b with Mbd4 or Tdg
A34V/K44Q/M66T/L214S/Y229H
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isolation of a relaxed-specificity mutant of the enzyme also methylates, at a lower rate, GG(G/C)CC sites
L214S/Y229H
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isolation of a relaxed-specificity mutant of the enzyme also methylates, at a lower rate, GG(G/C)CC sites
N172S
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isolation of a relaxed-specificity mutant of the enzyme also methylates, at a lower rate, GG(G/C)CC sites, the mutant enzyme displays enhanced protection of the cell DNA against the Sau96I endonuclease. The protection is not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the mutant
V173L
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isolation of a relaxed-specificity mutant of the enzyme also methylates, at a lower rate, GG(G/C)CC sites, the mutant enzyme displays enhanced protection of the cell DNA against the Sau96I endonuclease. The protection is not sufficient to support long-term survival in the presence of the inducer, which is consistent with incomplete methylation of GG(G/C)CC sites in plasmid DNA purified from the mutant
C141S
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site-directed mutagenesis in motif IV
C368A
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the mutation does not affect the enzyme activity
E186A
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site-directed mutagenesis in motif VI, almost inactive mutant
K297A
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site-directed mutagenesis in motif TRD, the mutant is resistant to Hin6I digestion
N299A
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site-directed mutagenesis in motif TRD, the mutant is resistant to Hin6I digestion
Q147L
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site-directed mutagenesis in motif IV, the mutant shows less than 10% of wild-type activity
R230A
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site-directed mutagenesis in motif VIII, almost inactive mutant
R232A
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site-directed mutagenesis in motif VIII, the mutant shows less than 10% of wild-type activity
S145A
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site-directed mutagenesis in motif IV, the mutant shows less than 10% of wild-type activity
S2C/C141S/C368A
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replacement of the active site Cys141 reduces the enzyme activity, while the substitutions Ser2Cys and Cys368Ala as well as a C-terminal Ser-His6 tag do not affect the enzyme activity
S317A
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site-directed mutagenesis in motif TRD, the mutant shows about 50% of wild-type activity
T313A
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site-directed mutagenesis in motif TRD, the mutant is resistant to Hin6I digestion
T313D
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site-directed mutagenesis in motif TRD, almost inactive mutant
T313H
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site-directed mutagenesis in motif TRD, almost inactive mutant
V188X
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replacement of the conserved Val188 from the ENV motif VI results in a 5fold increased DNA binding affinity and a 2fold decreased catalytic activity
additional information
CG methylation is lost in a drm1/2/cmt3 mutant, and non-CG methylation is almost completely eliminated in a met1 mutant. Exceptional maintenance of methylation at a CCmTGG site in some epigenetic mutants, overview
additional information
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CG methylation is lost in a drm1/2/cmt3 mutant, and non-CG methylation is almost completely eliminated in a met1 mutant. Exceptional maintenance of methylation at a CCmTGG site in some epigenetic mutants, overview
additional information
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enzyme overexpression does not cause an altered phenotype
additional information
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generation of a series of null mutations by remobilization of the EP(2)GE15695 element inserted 108 bp upstream of the Dnmt2 gene. EP(2)GE15695 does not affect the Dnmt2 gene. Dnmt2 null strains show stable loss of the subtelomeric clusters of defective Invader4 elements, phenotype, overview
additional information
construction of a z2389 null mutant
additional information
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construction of a z2389 null mutant
additional information
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removal of 50 amino acids from the N-terminus of the beta fragment of M.EcoHK31I severely reduces but does not eliminate methylation activity in vivo. Design of the methylation target site and fusion of the a and betaDELTA42 fragments to zinc fingers
additional information
construction of zinc-finger-domain point mutant and deletion mutant DNMT1 clones, CXXC deleted DNMT1, i.e. mutant DNMT1DELTACXXC and DNMT1 lacking the first 580 amino acids, i.e. DNMT1DELTA580. The latter prefers binding to unmethylated DNA instead of hemimethylated or fully methylated DNA like the wild-type enzyme. Deletion of CXXC domain in DNMT1 does not affect replication foci occupancy during DNA replication. A permanent cell line with DNMT1DELTACXXC displays partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme
additional information
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construction of zinc-finger-domain point mutant and deletion mutant DNMT1 clones, CXXC deleted DNMT1, i.e. mutant DNMT1DELTACXXC and DNMT1 lacking the first 580 amino acids, i.e. DNMT1DELTA580. The latter prefers binding to unmethylated DNA instead of hemimethylated or fully methylated DNA like the wild-type enzyme. Deletion of CXXC domain in DNMT1 does not affect replication foci occupancy during DNA replication. A permanent cell line with DNMT1DELTACXXC displays partial loss of genomic methylation on rDNA loci, despite the presence of endogenous wild-type enzyme
additional information
creation of genes encoding circularly permuted variants of M.MpeI. Eleven of the fourteen M.MpeI permutants have detectable MTase activity. Permutant cp62M.MpeI, in which the new N-terminus is located between conserved motifs II and III, has by far the highest activity, comparable to the activity of wild-type M.MpeI. The MTase activity of the permutants measured in cell extracts is in most cases substantially lower than that of the wild-type enzyme, although permutation sites were designed to fall outside of conserved motifs
additional information
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creation of genes encoding circularly permuted variants of M.MpeI. Eleven of the fourteen M.MpeI permutants have detectable MTase activity. Permutant cp62M.MpeI, in which the new N-terminus is located between conserved motifs II and III, has by far the highest activity, comparable to the activity of wild-type M.MpeI. The MTase activity of the permutants measured in cell extracts is in most cases substantially lower than that of the wild-type enzyme, although permutation sites were designed to fall outside of conserved motifs
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additional information
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construction of Dnmt1 mutants, two alleles of the endogenous Dnmt1 locus of R1 embryonic stem cells are sequentially modified using two similar targeting vectors to generate the DOX-OFF Dnmt1tet/tet cell line. The isolated 5' portion of the mouse Dnmt1 gene is used for targeted mutagenesis. Transient absence of DNMT1 expression leads to loss of imprinted methylation. In an embryonic stem cell line endogenous DNMT1s expression is extinguished by the addition of doxycycline, phenotypes, overview
additional information
construction of several Xpress-tagged Dnmt3b C-terminal truncation mutants, Dnmt3b mutants possessing the conserved MTase motifs VI, IV or I are all capable of binding Tdg, while removal of the entire catalytic domain of Dnmt3b disrupts its interaction with Tdg. Deletion of both putative interaction regions, PWWP and I, from Dnmt3b eliminates the binding ability. Reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression
additional information
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even modest increases or decreases in levels of m5C cause severe abnormalities or death in mutant mice
additional information
truncated inactive N-terminal fragments of M.SssI can assemble with truncated inactive C-terminal fragments to form active enzyme in vivo when produced in the same Escherichia coli cell. Overlapping and non-overlapping fragments as well as fragments containing short appended foreign sequences have complementation capacity. In optimal combinations C-terminal fragments start between conserved motif VIII. Fusions of two N-terminal and two C-terminal fragments to 21.6 kDa zinc finger domains only slightly reduce complementation ability of the fragments
additional information
creation of genes encoding circularly permuted variants of M.SssI. Six of the seven M.SssI permutants have detectable MTase activity. Permutant cp58M.SssI, in which the new N-terminus is located between conserved motifs II and III, has by far the highest activity. The MTase activity of the permutants measured in cell extracts is in most cases substantially lower than that of the wild-type enzyme, although permutation sites were designed to fall outside of conserved motifs
additional information
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truncated inactive N-terminal fragments of M.SssI can assemble with truncated inactive C-terminal fragments to form active enzyme in vivo when produced in the same Escherichia coli cell. Overlapping and non-overlapping fragments as well as fragments containing short appended foreign sequences have complementation capacity. In optimal combinations C-terminal fragments start between conserved motif VIII. Fusions of two N-terminal and two C-terminal fragments to 21.6 kDa zinc finger domains only slightly reduce complementation ability of the fragments
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additional information
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creation of genes encoding circularly permuted variants of M.SssI. Six of the seven M.SssI permutants have detectable MTase activity. Permutant cp58M.SssI, in which the new N-terminus is located between conserved motifs II and III, has by far the highest activity. The MTase activity of the permutants measured in cell extracts is in most cases substantially lower than that of the wild-type enzyme, although permutation sites were designed to fall outside of conserved motifs
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