Braguglia D, Heun P, Pasero P, Duncker B P, Gasser S M
Swiss Institute for Experimental Cancer Research, Ch. des Boveresses 155, Epalinges/Lausanne, CH-1066, Switzerland.
J Mol Biol. 1998 Aug 28;281(4):631-49. doi: 10.1006/jmbi.1998.1973.
We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.
我们描述了从同步于S期的酵母细胞中制备核提取物的方法，该提取物能在体外支持无引物、超螺旋质粒的对阿非迪霉素敏感的半保留复制。这通过对掺入了[α-32P]dATP的复制中间体进行一维和二维凝胶电泳、将甲基化模板DNA转化为半甲基化或抗DpnI形式以及用重衍生物BrdUTP替代dTTP来监测，后者会导致密度变化，对应于完整的第二条链合成。我们证明了其对DNA聚合酶δ和聚合酶α/引发酶复合物的依赖性，并且能够在ATP限制条件下检测到假定的冈崎片段。与超螺旋质粒的半保留复制不同，线性或开环模板通过修复合成掺入标记核苷酸，在CsCl梯度上不会产生明显的密度变化。与真正的复制反应一致，我们发现相对于G1期核提取物，质粒DNA的半保留复制在S期受到刺激，并且与促进重组的因子Rad52p无关。使用这个新系统，我们证明半保留复制而非聚合酶活性本身需要由DNA2编码的DNA解旋酶的活性。
