Tal M, Shimron F, Yagil G
Department of Cell Biology, Weizmann Institute of Science, Rehovet, Israel.
J Mol Biol. 1994 Oct 21;243(2):179-89. doi: 10.1006/jmbi.1994.1645.
The DNA of the centromere of chromosome IV (CEN4) of Saccharomyces cerevisiae is found to be sensitive to single-strand specific nuclease P1 when inserted into a negatively supercoiled plasmid. Fine mapping identifies two P1-sensitive segments: one segment maps to essential centromere element CDEI and bordering CDEII bases, and the other segment is located in element CDEIII. The AT-rich element CDEII, which is expected to be early melting, is for the most part resistant to nuclease P1. Cleavage is inhibited by NaCl, MgCl2 and polyamines. The cleavage rate is only weakly dependent on P1 concentration in the range of 0.5 to 20 munits/microliters. The two P1-sensitive segments are also modified by the DNA-confirmation-specific reagent KMnO4. Negative superhelicity is required for all modifications. Two-dimensional topoisomer analysis indicates the unwinding of 80(+/- 10) bases within the negatively supercoiled CEN4-containing plasmid. The data best fit a model in which the DNA of the CEN4 region undergoes a transition into a paranemic intermediate in which each strand is folded into an RNA-like foldback structure.
酿酒酵母第四条染色体（CEN4）着丝粒的DNA插入负超螺旋质粒后，被发现对单链特异性核酸酶P1敏感。精细定位确定了两个P1敏感片段：一个片段定位于必需着丝粒元件CDEI及相邻的CDEII碱基，另一个片段位于元件CDEIII中。富含AT的元件CDEII预计会较早解链，但其大部分对核酸酶P1具有抗性。NaCl、MgCl2和多胺可抑制切割。在0.5至20单位/微升的范围内，切割速率仅微弱依赖于P1浓度。这两个P1敏感片段也被DNA构象特异性试剂KMnO4修饰。所有修饰都需要负超螺旋。二维拓扑异构分析表明，含CEN4的负超螺旋质粒内有80（±10）个碱基解链。数据最符合一个模型，即CEN4区域的DNA转变为一种平行排列的中间体，其中每条链都折叠成类似RNA的回折结构。
