Krokan H, Bjorklid E, Prydz H
Biochemistry. 1975 Sep 23;14(19):4227-32. doi: 10.1021/bi00690a012.
DNA replication in isolated nuclei from synchronized HeLa cells has been studied in an effort to optimalize the system and characterize the product. The synthesis was highly dependent on the four deoxyribonucleoside triphosphates, ATP, and Mg2+. Optimum pH was about 7.8. The system was further stimulated by monovalent ions with NH4Cl and Tris-HCl (each 65 mM) being the most effective. The four ribonucleoside triphosphates and glycerol gave a slight but very reproducible and additive stimulation. Low concentrations of spermine and spermidine (0.2-1.5 X 10(-4) M) were also slightly stimulatory (10-15%) whereas higher concentrations were inhibitory. The reaction product was DNase sensitive, and banded at 1.699 g/ml in neutral CsCl together with bulk HeLa nuclear DNA. When studied by neutral CsCl and alkaline Cs2SO4 gradients, the incorporation of [3H]TTP was mainly (more than 85%) due to further elongation of strands initiated in vivo as evidenced by BrdUrd labeling.
为了优化系统并表征产物，对来自同步化HeLa细胞的分离细胞核中的DNA复制进行了研究。该合成高度依赖于四种脱氧核糖核苷三磷酸、ATP和Mg2+。最适pH约为7.8。单价离子能进一步刺激该系统，其中NH4Cl和Tris-HCl（均为65 mM）最为有效。四种核糖核苷三磷酸和甘油能产生轻微但非常可重复的累加刺激。低浓度的精胺和亚精胺（0.2 - 1.5×10(-4) M）也有轻微刺激作用（10 - 15%），而高浓度则有抑制作用。反应产物对DNase敏感，在中性CsCl中与大量HeLa核DNA一起在1.699 g/ml处形成条带。当通过中性CsCl和碱性Cs2SO4梯度进行研究时，[3H]TTP的掺入主要（超过85%）是由于体内起始链进一步延长，这由BrdUrd标记证明。
