ON THE FIDELITY OF DNA-REPLICATION - MANGANESE MUTAGENESIS INVITRO
ON THE FIDELITY OF DNA-REPLICATION - MANGANESE MUTAGENESIS INVITRO
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DOI：
10.1021/bi00342a019
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发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
LOEB, LA
中科院分区：
文献类型：
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作者：
BECKMAN, RA;MILDVAN, AS;LOEB, LA
在使用多种酶和模板的研究中，锰在体内和体外均具有致突变性。使用大肠杆菌DNA聚合酶I与聚[d（A-T）]和. X174 DNA模板，我们分析了锰诱变的机制，通过确定错误率对游离Mn 2+浓度的依赖性，并将其与测得的Mn 2+从酶，模板和脱氧核苷三磷酸底物的解离常数进行比较。这种比较表明了几个结论：（1）在非常低的Mn 2+浓度下，该酶以高保真度被激活。因此，锰本身的活化不太可能显著改变酶的构象，从而影响核苷酸选择。(2)在低游离Mn 2+浓度（< 100 μ M）下，锰通过其与DNA模板的相互作用引起掺入错误。诱变的浓度依赖性取决于Mn 2+与所用特定DNA模板的结合强度。这些数据不允许排除Mn 2 +-脱氧核苷三磷酸相互作用在选定情况下促成诱变的可能性。游离Mn 2+浓度的该范围是与体内诱变最相关的范围之一。(3)在更高浓度（500 μ M至1.5mM）下，发生Mn 2+的进一步诱变。这种诱变可能是由于锰结合到DNA内的单链区域或酶上的弱辅助位点。
Manganese is mutagenic in vivo and in vitro in studies with a variety of enzymes and templates. Using Escherichia coli DNA polymerase I with poly [d(A-T)] and .vphi. X174 DNA templates, we analyzed the mechanism of manganese mutagenesis by determining the dependence of error rate on free Mn2+ concentration and comparing this to measured dissociation constants of Mn2+ from enzyme, template, and deoxynucleoside triphosphate substrates. This comparison suggests several conclusions: (1) At very low Mn2+ concentrations, the enzyme is activated at high fidelity. Thus, it is unlikely that activation with manganese per se significantly alters the conformation of the enzyme so as to affect nucleotide selection. (2) At low free Mn2+ concentrations (< 100 .mu.M), manganese causes errors in incorporation via its interaction with the DNA template. The concentration dependence of mutagenesis is determined by the strength of binding Mn2+ to the particular DNA template used. The data do not allow one to rule out the possibility that Mn2+-deoxynucleoside triphosphate interactions contribute to mutagenesis in selected situations. This range of free Mn2+ concentrations is the one of greatest relevance for in vivo mutagenesis. (3) At higher concentrations (between 500 .mu.M and 1.5 mM), further mutagenesis by Mn2+ occurs. This mutagenesis probably is due either to binding of manganese to single-stranded regions within the DNA or to weak accessory sites on the enzyme.