Source:http://linkedlifedata.com/resource/pubmed/id/10551868
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
46
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| pubmed:dateCreated |
2000-1-3
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| pubmed:abstractText |
A method is described to express and purify human DNA (cytosine-5) methyltransferase (human DNMT1) using a protein splicing (intein) fusion partner in a baculovirus expression vector. The system produces approximately 1 mg of intact recombinant enzyme >95% pure per 1.5 x 10(9) insect cells. The protein lacks any affinity tag and is identical to the native enzyme except for the two C-terminal amino acids, proline and glycine, that were substituted for lysine and aspartic acid for optimal cleavage from the intein affinity tag. Human DNMT1 was used for steady-state kinetic analysis with poly(dI-dC).poly(dI-dC) and unmethylated and hemimethylated 36- and 75-mer oligonucleotides. The turnover number (k(cat)) was 131-237 h(-1) on poly(dI-dC).poly(dI-dC), 1.2-2.3 h(-1) on unmethylated DNA, and 8.3-49 h(-1) on hemimethylated DNA. The Michaelis constants for DNA (K(m)(CG)) and S-adenosyl-L-methionine (AdoMet) (K(m)(AdoMet)) ranged from 0.33-1.32 and 2.6-7.2 microM, respectively, whereas the ratio of k(cat)/K(m)(CG) ranged from 3.9 to 44 (237-336 for poly(dI-dC).poly(dI-dC)) x 10(6) M(-1) h(-1). The preference of the enzyme for hemimethylated, over unmethylated, DNA was 7-21-fold. The values of k(cat) on hemimethylated DNAs showed a 2-3-fold difference, depending upon which strand was pre-methylated. Furthermore, human DNMT1 formed covalent complexes with substrates containing 5-fluoro-CNG, indicating that substrate specificity extended beyond the canonical CG dinucleotide. These results show that, in addition to maintenance methylation, human DNMT1 may also carry out de novo and non-CG methyltransferase activities in vivo.
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| pubmed:grant | |
| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/DNA (Cytosine-5-)-Methyltransferase,
http://linkedlifedata.com/resource/pubmed/chemical/FMR1 protein, human,
http://linkedlifedata.com/resource/pubmed/chemical/Fmr1 protein, mouse,
http://linkedlifedata.com/resource/pubmed/chemical/Fragile X Mental Retardation Protein,
http://linkedlifedata.com/resource/pubmed/chemical/Nerve Tissue Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Oligodeoxyribonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/Polydeoxyribonucleotides,
http://linkedlifedata.com/resource/pubmed/chemical/RNA-Binding Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Recombinant Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Ribonucleoproteins, Small Nuclear,
http://linkedlifedata.com/resource/pubmed/chemical/S-Adenosylmethionine,
http://linkedlifedata.com/resource/pubmed/chemical/poly d(I-C)
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| pubmed:status |
MEDLINE
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| pubmed:month |
Nov
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| pubmed:issn |
0021-9258
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
12
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| pubmed:volume |
274
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
33002-10
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| pubmed:dateRevised |
2007-11-14
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| pubmed:meshHeading |
pubmed-meshheading:10551868-Animals,
pubmed-meshheading:10551868-Baculoviridae,
pubmed-meshheading:10551868-DNA (Cytosine-5-)-Methyltransferase,
pubmed-meshheading:10551868-DNA Methylation,
pubmed-meshheading:10551868-Fragile X Mental Retardation Protein,
pubmed-meshheading:10551868-Humans,
pubmed-meshheading:10551868-Kinetics,
pubmed-meshheading:10551868-Mice,
pubmed-meshheading:10551868-Nerve Tissue Proteins,
pubmed-meshheading:10551868-Oligodeoxyribonucleotides,
pubmed-meshheading:10551868-Polydeoxyribonucleotides,
pubmed-meshheading:10551868-Protein Splicing,
pubmed-meshheading:10551868-RNA-Binding Proteins,
pubmed-meshheading:10551868-Recombinant Proteins,
pubmed-meshheading:10551868-Ribonucleoproteins, Small Nuclear,
pubmed-meshheading:10551868-S-Adenosylmethionine,
pubmed-meshheading:10551868-Spodoptera
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| pubmed:year |
1999
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| pubmed:articleTitle |
Recombinant human DNA (cytosine-5) methyltransferase. I. Expression, purification, and comparison of de novo and maintenance methylation.
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| pubmed:affiliation |
New England Biolabs, Beverly, Massachusetts 01915, USA.
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| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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