Pasero P, Braguglia D, Gasser S M
Swiss Institute for Experimental Cancer Research, Epalinges/Lausanne.
Genes Dev. 1997 Jun 15;11(12):1504-18. doi: 10.1101/gad.11.12.1504.
We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro.
我们描述了一种来自酵母的体外复制分析方法，其中将完整的细胞核添加到S期核提取物中会导致脱氧核苷酸在空间上离散的位点掺入基因组DNA。当用BrdUTP替代dTTP时，新合成的部分DNA在CsCl梯度上的密度发生变化，这表明是半保留复制。起始以起源特异性方式发生，并且可以在G1期或S期细胞核中检测到，但在G2期或有丝分裂细胞核中则检测不到。S期提取物含有促进G1期细胞核复制所必需的对热和6-DMAP敏感的成分。在orc2-1突变体中，核DNA的复制在限制温度下被阻断，并且无活性的Orc2p不能被含有野生型ORC的提取物反式互补。在G1期被阻断的cln缺陷型细胞核中DNA复制的起始表明，ORC依赖性复制前复合体在Start之前形成。这代表了第一个非病毒和非胚胎复制系统，其中DNA复制在体外以ORC依赖性和起源特异性方式起始。
