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 (m(3)A), 7-methylguanine(m(7)G), and 3-methylguanine (m(3)G) from DNA, and the K-m and k(cat) values were 130 nM and 0.8 min(-1) for m(3)A, and 860 nM and 0.2 min(-1) for m(7)G, respectively, when methylated calf thymus DNA was used as the substrate. A comparison of k(cat)/K-m values for different bases indicates that the enzyme was more efficient in excising both m(3)A and m(3)G than m(7)G from methylated DNA. The enzyme showed moderate binding affinities (K-A) 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 E(1%)(280nm) 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 m(3)A and m(7)G 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 m(3)A or m(7)G. However, 1.0 mM m(3)A stimulated release of m(7)G. 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.