Gautschi J R, Burkhalter M, Baumann E A
Biochim Biophys Acta. 1978 Mar 29;518(1):31-6. doi: 10.1016/0005-2787(78)90113-2.
Relative utilization, of dThd, BrdUrd, and IdUrd as exogenous precursors of DNA in vivo was compared with utilization of the respective triphosphates in vitro. To measure relative utilization, exponentially growing P-815 or CHO cells were incubated with different mixtures of dThd/BrdUrd or dThd/IdUrd in the presence of FdUrd and dCyd, and Brij-58 lysed cells with mixtures of dTTP/BrdUTP or dTTP/IdUTP. The degree of substitution of dTMP by BrdUMP or IdUMP in DNA newly synthesized in vivo or in vitro was determined by isopycnic centrifugation. BrdUrd was utilized by intact cells with equal efficiency as dThd, while IdUrd was less efficiently incorporated when the total concentration of dThd and the tested analogue was 3.10(-5) M. During semiconservative DNA replication in vitro, however, dTTP, BrdUTP and IdUTP were utilized with equal efficiency. Thus, we conclude that observed preference for dThd by intact cells incubated with dThd/IdUrd mixtures is related to characteristics of precursor metabolism and not to different affinities of the replication complex for the respective precursors. dUTP was rapidly dephosphorylated in vitro and no incorporation into DNA was detected.
将体内dThd、BrdUrd和IdUrd作为DNA外源前体的相对利用率与相应三磷酸酯在体外的利用率进行了比较。为了测量相对利用率，在FdUrd和dCyd存在的情况下，将指数生长的P-815或CHO细胞与dThd/BrdUrd或dThd/IdUrd的不同混合物一起孵育，并用dTTP/BrdUTP或dTTP/IdUTP混合物裂解细胞。通过等密度离心法测定体内或体外新合成的DNA中dTMP被BrdUMP或IdUMP取代的程度。完整细胞对BrdUrd的利用率与dThd相同，而当dThd和受试类似物的总浓度为3×10⁻⁵ M时，IdUrd的掺入效率较低。然而，在体外半保留DNA复制过程中，dTTP、BrdUTP和IdUTP的利用率相同。因此，我们得出结论，用dThd/IdUrd混合物孵育的完整细胞对dThd的偏好与前体代谢的特性有关，而与复制复合物对相应前体的不同亲和力无关。dUTP在体外迅速脱磷酸化，未检测到其掺入DNA。
