Estes H G, Robinson B S, Eisenberg S
Department of Microbiology, School of Medicine, University of Connecticut Health Center, Farmington 06030.
Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11156-60. doi: 10.1073/pnas.89.23.11156.
We have reconstituted in vitro a multistage assembly of a protein complex that specifically recognizes a yeast genomic origin of replication, the autonomously replicating sequence ARS121. The first step in the assembly was the interaction of the known origin-binding factor OBF1 and another factor, OBF2, with the ARS121 origin of replication to form the OBF1-OBF2-origin complex. This complex was the substrate for the ATP-dependent binding of a third DNA-binding activity, the core binding factor, CBF. Binding of CBF to the origin, identified by the retarded mobility of the origin DNA fragment in agarose gels, required, in addition to ATP and the OBF1-OBF2-origin complex, a functional essential core nucleotide sequence. ARS121 DNA containing mutations in the core, which inactivate the origin in vivo, did not sustain stable CBF binding, whereas ARS121 DNA mutated outside the boundaries of the essential core, which has normal origin function, bound CBF as wild type. This tight, direct correlation between the ability of the origin to bind CBF and its function as an origin of replication in vivo strongly suggest that the multiprotein complex reconstituted in vitro has a key role in the initiation of DNA replication.
我们在体外重建了一种蛋白质复合物的多阶段组装过程，该复合物能特异性识别酵母基因组复制起点——自主复制序列ARS121。组装的第一步是已知的起点结合因子OBF1和另一个因子OBF2与ARS121复制起点相互作用，形成OBF1 - OBF2 - 起点复合物。这个复合物是第三种DNA结合活性物质——核心结合因子CBF进行ATP依赖性结合的底物。通过琼脂糖凝胶中起点DNA片段迁移率的降低来确定，CBF与起点的结合除了需要ATP和OBF1 - OBF2 - 起点复合物外，还需要一个功能性的必需核心核苷酸序列。核心区域含有突变的ARS121 DNA在体内会使起点失活，无法维持CBF的稳定结合，而在必需核心边界之外发生突变但具有正常起点功能的ARS121 DNA则能像野生型一样结合CBF。起点结合CBF的能力与其在体内作为复制起点的功能之间这种紧密、直接的相关性，强烈表明在体外重建的多蛋白复合物在DNA复制起始过程中起关键作用。
