Rashid M B, Shirahige K, Ogasawara N, Yoshikawa H
Department of Genetics, Osaka University Medical School, Suita, Japan.
Gene. 1994 Dec 15;150(2):213-20. doi: 10.1016/0378-1119(94)90429-4.
We have analyzed the relationship between autonomously replicating sequence (ARS) structure and function for three ARS (ARS605, ARS607 and ARS609) from chromosome VI of Saccharomyces cerevisiae by systematic XhoI-linker mutation in the ARS consensus sequence (ACS) and flanking sequences. All mutations that encroached upon the ACS destroyed ARS activity. DNA sequences stimulative for ARS function were identified on either side of the ACS of ARS605 and only on the 3'-side of the ACS of ARS607. In ARS609, however, no such stimulative sequences were observed. Base substitutions complementary to the wild-type sequence of those stimulative regions, in ARS605 and ARS607, that did not change the delta G of unwinding nor affected ARS activity suggests that these regions have, at least, a function as DNA-unwinding elements (DUE). ARS605, ARS607 and ARS609 DNA are of low delta G value and showed hypersensitivity to single-strand-specific nuclease when inserted in negatively supercoiled plasmid. Linker mutations inhibitory for ARS activity (5L11 and 7L14) also caused significant changes in local nucleotide (nt) sensitivity within the ACS and its adjoining regions. Complementary base substitutions, however, did not affect these changes in local nt sensitivity. These results imply that the stimulative regions flanking the ACS are necessary to produce an optimum conformation around the ACS which may be important for full ARS activity.
我们通过对酿酒酵母第六条染色体上的三个自主复制序列（ARS，即ARS605、ARS607和ARS609）的ARS共有序列（ACS）及其侧翼序列进行系统性的XhoI接头突变，分析了ARS结构与功能之间的关系。所有侵入ACS的突变都会破坏ARS活性。在ARS605的ACS两侧均鉴定出了对ARS功能有刺激作用的DNA序列，而在ARS607的ACS中，仅在其3'端一侧发现了此类序列。然而，在ARS609中未观察到此类刺激序列。在ARS605和ARS607中，那些与刺激区域的野生型序列互补、既不改变解链的ΔG也不影响ARS活性的碱基替换表明，这些区域至少具有作为DNA解链元件（DUE）的功能。ARS605、ARS607和ARS609的DNA具有较低的ΔG值，当插入负超螺旋质粒时，对单链特异性核酸酶表现出超敏感性。抑制ARS活性的接头突变（5L11和7L14）也导致了ACS及其相邻区域内局部核苷酸（nt）敏感性的显著变化。然而，互补碱基替换并未影响局部nt敏感性的这些变化。这些结果表明，ACS侧翼的刺激区域对于在ACS周围产生最佳构象是必要的，这对于完整的ARS活性可能很重要。
