Purification and biochemical characterization of recombinant N-methylpurine-DNA glycosylase of the mouse.
The mouse N-methylpurine-DNA glycosylase (MPG), responsible for the removal of most N-alkyladducts in DNA, was purified to homogeneity as a recombinant nonfusion protein from Escherichia coli. Only 10-15% of the protein was present in the soluble form in E. coli cells. The N-terminal amino acid sequence of the purified protein which lacks 48 residues from the amino terminus of the wild type protein was identical to that predicted from the nucleotide sequence. The glycosylase hydrolyzes 3-methyladenine (m3A), 7-methylguanine(m7G), and 3-methylguanine (m3G) from DNA, and the Km and kcat values were 130 nM and 0.8 min-1 for m3A, and 860 nM and 0.2 min-1 for m7G, respectively, when methylated calf thymus DNA was used as the substrate. A comparison of kcat/Km values for different bases indicates that the enzyme was more efficient in excising both m3A and m3G than m7G from methylated DNA. The enzyme showed moderate binding affinities (KA) for both methylated (5.8 x 10(7) M-1) and nonmethylated DNAs (4.2 x 10(7) M-1). The mouse protein has an extinction coefficient E280nm1% of 10.5 and a pI of 9.3. The enzyme activity was optimal in the presence of 100 mM NaCl, with a broad pH optimum of 8.5-9.5. The enzymatic release of both m3A and m7G was stimulated 50-75% by 0.5 mM MgCl2 and 0.02 mM spermine but inhibited by higher concentrations of these agents. Product inhibition by 40-50% of the reaction occurred in the presence of 10 mM m3A or m7G. However, 1.0 mM m3A stimulated release of m7G. The enzyme was inhibited by 60% in the presence of 0.9 mg/mL DNA which, at the same time, protected it from thermal inactivation.
Roy
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Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555.
Brooks
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Mitra
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D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D013379:Substrate Specificity
D011994:Recombinant Proteins; D015636:Magnesium Chloride; C044502:3-methylguanine; D013096:Spermine; D012965:Sodium Chloride; C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II; D000225:Adenine