PMID- 10551868
OWN - NLM
STAT- MEDLINE
DCOM- 20000103
LR  - 20190508
IS  - 0021-9258 (Print)
IS  - 0021-9258 (Linking)
VI  - 274
IP  - 46
DP  - 1999 Nov 12
TI  - Recombinant human DNA (cytosine-5) methyltransferase. I. Expression,
      purification, and comparison of de novo and maintenance methylation.
PG  - 33002-10
AB  - 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.
FAU - Pradhan, S
AU  - Pradhan S
AD  - New England Biolabs, Beverly, Massachusetts 01915, USA.
FAU - Bacolla, A
AU  - Bacolla A
FAU - Wells, R D
AU  - Wells RD
FAU - Roberts, R J
AU  - Roberts RJ
LA  - eng
GR  - GM46127/GM/NIGMS NIH HHS/United States
GR  - GM52982/GM/NIGMS NIH HHS/United States
GR  - NS37554/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - J Biol Chem
JT  - The Journal of biological chemistry
JID - 2985121R
RN  - 0 (FMR1 protein, human)
RN  - 0 (Fmr1 protein, mouse)
RN  - 0 (Nerve Tissue Proteins)
RN  - 0 (Oligodeoxyribonucleotides)
RN  - 0 (Polydeoxyribonucleotides)
RN  - 0 (RNA-Binding Proteins)
RN  - 0 (Recombinant Proteins)
RN  - 0 (Ribonucleoproteins, Small Nuclear)
RN  - 139135-51-6 (Fragile X Mental Retardation Protein)
RN  - 25853-45-6 (poly d(I-C))
RN  - 7LP2MPO46S (S-Adenosylmethionine)
RN  - EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferase 1)
RN  - EC 2.1.1.37 (DNA (Cytosine-5-)-Methyltransferases)
RN  - EC 2.1.1.37 (DNMT1 protein, human)
RN  - EC 2.1.1.37 (Dnmt1 protein, mouse)
SB  - IM
MH  - Animals
MH  - Baculoviridae/genetics
MH  - DNA (Cytosine-5-)-Methyltransferase 1
MH  - DNA (Cytosine-5-)-Methyltransferases/chemistry/*genetics
MH  - *DNA Methylation
MH  - Fragile X Mental Retardation Protein
MH  - Humans
MH  - Kinetics
MH  - Mice
MH  - Nerve Tissue Proteins/genetics
MH  - Oligodeoxyribonucleotides/metabolism
MH  - Polydeoxyribonucleotides/metabolism
MH  - Protein Splicing
MH  - *RNA-Binding Proteins
MH  - Recombinant Proteins/chemistry/genetics
MH  - Ribonucleoproteins, Small Nuclear/genetics
MH  - S-Adenosylmethionine/metabolism
MH  - Spodoptera/genetics
EDAT- 1999/11/07 00:00
MHDA- 1999/11/07 00:01
CRDT- 1999/11/07 00:00
PHST- 1999/11/07 00:00 [pubmed]
PHST- 1999/11/07 00:01 [medline]
PHST- 1999/11/07 00:00 [entrez]
AID - 10.1074/jbc.274.46.33002 [doi]
PST - ppublish
SO  - J Biol Chem. 1999 Nov 12;274(46):33002-10. doi: 10.1074/jbc.274.46.33002.