Kinetic and DNA-binding properties of recombinant human O6-methylguanine-DNA methyltransferase.
O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that plays an important role in chemotherapy, mutagenesis, and carcinogenesis. Recombinant human MGMT was isolated from an Escherichia coli high performance expression system and purified to homogeneity. The kinetic and DNA-binding properties of the recombinant human MGMT were studied. The purified human MGMT reacted stoichiometrically with methylated DNA under second-order rate kinetics. The rate constant with normal methylated DNA was 1 x 10(9) M-1 min-1 at 37 degrees C. The binding to DNA was the rate determining step in the repair process. Approximately eight base pairs of the DNA substrate were covered by the human MGMT protein. The affinity constant for interaction of DNA to MGMT was approximately 4.7 x 10(5) M-1. The binding to methylated DNA was also examined; the binding affinity to methylated DNA was two times higher than that to unmodified DNA. The interaction with DNA induced a conformational change in the human MGMT protein as monitored by circular dichroism and fluorescence analysis. A similar conformational change was induced by both methylated and unmodified DNA.
Chan
C L
CL
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755-3835.
Wu
Z
Z
Ciardelli
T
T
Eastman
A
A
Bresnick
E
E
D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019853:O(6)-Methylguanine-DNA Methyltransferase; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D004273:DNA, Neoplasm; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase