Fox C A, Loo S, Dillin A, Rine J
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Genes Dev. 1995 Apr 15;9(8):911-24. doi: 10.1101/gad.9.8.911.
The role of the origin recognition complex (ORC) was investigated in replication initiation and in silencing. Temperature-sensitive mutations in ORC genes caused defects in replication initiation at chromosomal origins of replication, as measured by two-dimensional (2-D) origin-mapping gels, fork migration analysis, and plasmid replication studies. These data were consistent with ORC functioning as a eukaryotic replication initiator. Some origins displayed greater replication initiation deficiencies in orc mutants than did others, revealing functional differences between origins. Alleles of ORC5 were isolated that were defective for silencing but not replication, indicating that ORC's role in silencing could be separated from its role in replication. In temperature-sensitive orc mutants arrested in mitosis, temperature-shift experiments caused a loss of silencing, indicating both that ORC had functions outside of the S phase of the cell cycle and that ORC was required for the maintenance of the silenced state.
研究了复制起始复合物(ORC)在复制起始和沉默中的作用。通过二维(2-D)起始点定位凝胶、叉迁移分析和质粒复制研究发现,ORC基因中的温度敏感突变导致染色体复制起始点的复制起始缺陷。这些数据与ORC作为真核生物复制起始因子的功能一致。一些起始点在orc突变体中表现出比其他起始点更大的复制起始缺陷,揭示了起始点之间的功能差异。分离出了ORC5的等位基因,这些等位基因在沉默方面有缺陷,但在复制方面没有缺陷,这表明ORC在沉默中的作用可以与其在复制中的作用分开。在有丝分裂中停滞的温度敏感orc突变体中,温度转换实验导致沉默丧失,这表明ORC在细胞周期的S期之外也有功能,并且ORC是维持沉默状态所必需的。