Umek R M, Kowalski D
Molecular and Cellular Biology Department, Roswell Park Memorial Institute, Buffalo, New York 14263.
Cell. 1988 Feb 26;52(4):559-67. doi: 10.1016/0092-8674(88)90469-2.
We have localized the DNA sequence that facilitates unwinding of a yeast replication origin, the H4 ARS. The readily unwound sequence lies adjacent to the previously characterized consensus core sequence of the ARS. Unwinding is detected through the formation of a single-strand-specific nuclease hypersensitive site in H4 ARS mutant derivatives present on supercoiled plasmids. Linker-scanning and linker-deletion derivatives exhibit wild-type nuclease hypersensitivity and ARS function, while large external deletions reduce or eliminate nuclease detectable unwinding and origin function. ARS unwinding and origin function can be rescued in the deletion mutants by inserting a biologically unrelated sequence with DNA unwinding properties similar to a functional ARS. The data clarify the nature of DNA sequence requirements in the ARS by suggesting that small substitutions, insertions, and deletions are tolerated in the region flanking the consensus core sequence because they do not significantly alter the unwinding properties of the region.
我们已经定位了促进酵母复制起点H4 ARS解旋的DNA序列。易于解旋的序列位于ARS先前已鉴定的共有核心序列附近。通过在超螺旋质粒上存在的H4 ARS突变衍生物中形成单链特异性核酸酶超敏位点来检测解旋。接头扫描和接头缺失衍生物表现出野生型核酸酶超敏性和ARS功能，而大的外部缺失则减少或消除了核酸酶可检测到的解旋和起点功能。通过插入具有与功能性ARS相似的DNA解旋特性的生物学上不相关的序列，可以在缺失突变体中挽救ARS解旋和起点功能。这些数据通过表明在共有核心序列侧翼区域允许小的替换、插入和缺失，因为它们不会显著改变该区域的解旋特性，从而阐明了ARS中DNA序列要求的性质。
