Sinha N K, Haimes M D
J Biol Chem. 1981 Oct 25;256(20):10671-83.
The proteins coded by bacteriophate T4 replication genes 32, 41, 43, 44, 45, 61, and 62 together can replicate phi X174 DNA templates very efficiently. The fidelity of this in vitro replication reaction has been measured using an infectivity assay. The product molecules have the same specific infectivity as the template DNA. When an amber mutant DNA template is used, no increase in the frequency of revertants is seen even after more than 60 duplications in vitro. By using imbalances in the concentrations of deoxynucleotide substrates, the error rate during DNA replication in vitro can be greatly increased. Control experiments indicate that the increased mutagenesis is not due to the presence of dITP or dUTP as contaminants in the deoxynucleotide substrates used. The increase in the frequency of revertants is linearly related to the ratio of the correct and the incorrect deoxynucleotides. Determination of the DNA sequence of the revertants induced shows that a change in DNA sequence of the amber site predicted from the nucleotide bias occurs. DNA synthesis in vitro resembles in vivo replication in that the error rate depends not only upon the base change required for reversion but also upon the neighboring DNA sequences. The error rate is estimated to be 5 X 10(-6) at am3 site, 6.4 X 10(-7) at am86 site, and less than 2.9 X 10(-7) at am9 site. Comparison of the frequency of G-T and A-C mispairs reveals that most AT leads to GC transition mutations occur through G-T mispairs. Measurement of the frequency of the mispairs required to induce transversion mutations reveals that these occur primarily through purine-purine mispairs. Transition mutations are more frequent than transversion mutations at both the am3 and the am86 sites. These observations support the models for base pairing errors proposed by Watson and Crick ((1953) Nature 171, 964-967) and Topal and Fresco ((1976) Nature 263, 285-289).
由噬菌体T4复制基因32、41、43、44、45、61和62共同编码的蛋白质能够非常高效地复制φX174 DNA模板。已使用感染性测定法测量了这种体外复制反应的保真度。产物分子与模板DNA具有相同的比感染性。当使用琥珀突变体DNA模板时，即使在体外进行60多次复制后，回复突变体的频率也未见增加。通过使用脱氧核苷酸底物浓度的不平衡，可以大大提高体外DNA复制过程中的错误率。对照实验表明，诱变率的增加并非由于所用脱氧核苷酸底物中存在作为污染物的dITP或dUTP。回复突变体频率的增加与正确和错误脱氧核苷酸的比例呈线性相关。对诱导产生的回复突变体的DNA序列测定表明，根据核苷酸偏差预测的琥珀位点的DNA序列发生了变化。体外DNA合成与体内复制相似，因为错误率不仅取决于回复所需的碱基变化，还取决于相邻的DNA序列。估计在am3位点的错误率为5×10⁻⁶，在am86位点为6.4×10⁻⁷，在am9位点小于2.9×10⁻⁷。对G-T和A-C错配频率的比较表明，大多数导致GC转换突变的AT是通过G-T错配发生的。对诱导颠换突变所需错配频率的测量表明，这些主要通过嘌呤-嘌呤错配发生。在am3和am86位点，转换突变比颠换突变更频繁。这些观察结果支持了沃森和克里克（（1953年）《自然》171, 964 - 967）以及托帕尔和弗雷斯科（（1976年）《自然》263, 285 - 289）提出的碱基配对错误模型。
