Baculovirus-mediated expression and characterization of the full-length murine DNA methyltransferase.
The original cDNA sequence reported for the murine DNA methyltransferase (MTase) was not full length. Recently, additional cDNA sequences have been reported that lie upstream of the original and contain an extended open reading frame with three additional ATGs in frame with the coding region [Tucker et al . (1996) Proc. Natl. Acad. Sci. USA , 93, 12920-12925; Yoder et al . (1996) J. Biol. Chem . 271, 31092-31097]. Genomic DNA upstream of this ATG contains two more ATGs in frame and no obvious splice site. We have constructed, and expressed in baculovirus, MTase clones that begin at each of these four ATGs and examined their properties. Constructs beginning with any of the first three ATGs as their initiator methionines give a predominant DNA MTase band of approximately 185 kDa on SDS-PAGE corresponding to translational initiation at the third ATG. The fourth ATG construct gives a much smaller protein band of 173 kDa. The 185 kDa protein was purified by HPLC, characterized by mass spectrometry and has a measured molecular mass of 184 +/- 0.5 kDa. All of these MTases were functional in vitro and steady state kinetic analysis showed that the recombinant proteins exhibit similar kinetic properties irrespective of their length. The homogeneous recombinant enzyme from the fourth ATG construct shows a 2.5-fold preference for a hemi-methylated DNA substrate as compared to an unmethylated substrate, whereas the 185 kDa protein is equally active on both substrates. The kinetic properties of the recombinant enzyme are similar to those reported for the native MTase derived from murine erythroleukemia cells. The new clones are capable of yielding large quantities of intact MTases for further structural and functional studies.
Pradhan
S
S
New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA.
Talbot
D
D
Sha
M
M
Benner
J
J
Hornstra
L
L
Li
E
E
Jaenisch
R
R
Roberts
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded; D015870:Gene Expression; D005822:Genetic Vectors; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D015254:DNA Modification Methylases