Huang R Y, Kowalski D
Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, NY 14263.
EMBO J. 1993 Dec;12(12):4521-31. doi: 10.1002/j.1460-2075.1993.tb06141.x.
We have defined a replication origin, ORI305, within chromosome III of Saccharomyces cerevisiae by means of mutational analysis. cis-acting elements required for origin activity in the chromosome, as assayed by two-dimensional gel electrophoresis of replication intermediates, are the same as those required for the function of an autonomously replicating sequence, ARS305, in a plasmid. Essential elements include (i) an 11 bp sequence that is a near match to the ARS consensus and (ii) a broad sequence directly 3' to the consensus near match. Origin function is inactivated by point mutations in the essential near match sequence, suggesting that the sequence contributes to specifying the origin in the chromosome. Other consensus near matches with different sequences are present but are not required. The essential 3'-flanking sequence exhibits DNA helical instability and is sensitive to deletion mutations that stabilize the DNA helix. The wild-type 3'-flanking sequence can be functionally substituted by dissimilar sequences that also exhibit helical instability. The requirement for DNA helical instability indicates that the essential 3'-flanking sequence serves as a DNA unwinding element in the chromosome.
我们通过突变分析在酿酒酵母的第三条染色体中定义了一个复制起点ORI305。通过对复制中间体进行二维凝胶电泳分析，发现染色体中起点活性所需的顺式作用元件与质粒中自主复制序列ARS305功能所需的元件相同。必需元件包括：（i）一段11bp的序列，它与ARS共有序列近乎匹配；（ii）一个直接位于共有序列近乎匹配处3'端的宽泛序列。起点功能会因必需的近乎匹配序列中的点突变而失活，这表明该序列有助于在染色体中确定起点。存在其他具有不同序列的共有序列近乎匹配，但并非必需。必需的3'侧翼序列表现出DNA螺旋不稳定性，并且对稳定DNA螺旋的缺失突变敏感。野生型3'侧翼序列可被同样表现出螺旋不稳定性的不同序列进行功能替代。对DNA螺旋不稳定性的要求表明，必需的3'侧翼序列在染色体中作为DNA解旋元件发挥作用。
