Zak P, Kleibl K, Laval F
Groupe Radiochimie de l'ADN, Unité 247, Institut Gustave Roussy, Villejuif, France.
J Biol Chem. 1994 Jan 7;269(1):730-3.
In order to compare the ability of the human and rat O6-methylguanine-DNA methyltransferases (transferases) to repair in vitro O6-methylguanine (O6-MeGua) and O4-methylthymine (O4-MeThy) residues, which are two mutagenic DNA adducts formed by alkylating agents, we have purified both proteins to homogeneity. Gel electrophoresis of the proteins shows that the O4-MeThy repair is due to the transfer of the methyl group from the alkylated base to the transferase molecules. However, both proteins repair with different efficiencies the O6-MeGua and O4-MeThy residues present in alkylated DNA, poly[d(G.C)], poly(dG.dC), or in alkylated poly[d(A.T)] and poly(dA.dT), respectively. Reaction of both proteins with either methylated residues follows a second-order kinetics. The rate constants are 1 x 10(9) M-1 min-1 for both proteins acting on O6-MeGua and 4.8 x 10(6) or 1.8 x 10(5) M-1 min-1 for the rat or human protein acting on O4-MeThy, respectively. The activity of the mammalian transferases on O4-MeThy present in a poly(dA.dT) substrate is inhibited by double-stranded DNA.
为了比较人和大鼠的O6-甲基鸟嘌呤-DNA甲基转移酶（转移酶）在体外修复O6-甲基鸟嘌呤（O6-MeGua）和O4-甲基胸腺嘧啶（O4-MeThy）残基的能力，这两种诱变DNA加合物是由烷基化剂形成的，我们已将这两种蛋白质纯化至同质。蛋白质的凝胶电泳表明，O4-MeThy的修复是由于甲基从烷基化碱基转移到转移酶分子上。然而，这两种蛋白质分别以不同的效率修复烷基化DNA、聚[d（G.C）]、聚（dG.dC）中存在的O6-MeGua和O4-MeThy残基，或烷基化的聚[d（A.T）]和聚（dA.dT）中存在的O6-MeGua和O4-MeThy残基。这两种蛋白质与甲基化残基的反应均遵循二级动力学。两种蛋白质作用于O6-MeGua的速率常数均为1×10⁹ M⁻¹ min⁻¹，大鼠或人蛋白质作用于O4-MeThy的速率常数分别为4.8×10⁶或1.8×10⁵ M⁻¹ min⁻¹。双链DNA可抑制哺乳动物转移酶对聚（dA.dT）底物中存在的O4-MeThy的活性。
