Miki Kensuke, Shimizu Mitsuhiro, Fujii Michihiko, Hossain Mohammad Nazir, Ayusawa Dai
International Graduate School of Arts and Sciences, Yokohama City University, Seto 22-2, Kanazawa-Ku, Yokohama, Kanagawa 236-0027, Japan.
Biochem Biophys Res Commun. 2008 Apr 11;368(3):662-9. doi: 10.1016/j.bbrc.2008.01.149. Epub 2008 Feb 5.
5-Bromodeoxyuridine (BrdU) modulates expression of particular genes associated with cellular differentiation and senescence. Our previous studies have suggested an involvement of chromatin structure in this phenomenon. Here, we examined the effect of 5-bromouracil on nucleosome positioning in vivo using TALS plasmid in yeast cells. This plasmid can stably and precisely be assembled nucleosomes aided by the alpha2 repressor complex bound to its alpha2 operator. Insertion of AT-rich sequences into a site near the operator destabilized nucleosome positioning dependent on their length and sequences. Addition of BrdU almost completely disrupted nucleosome positioning through specific AT-tracts. The effective AT-rich sequences migrated faster on polyacrylamide gel electrophoresis, and their mobility was further accelerated by substitution of thymine with 5-bromouracil. Since this property is indicative of a rigid conformation of DNA, our results suggest that 5-bromouracil disrupts nucleosome positioning by inducing A-form-like DNA.
5-溴脱氧尿苷（BrdU）可调节与细胞分化和衰老相关的特定基因的表达。我们之前的研究表明染色质结构参与了这一现象。在此，我们使用酵母细胞中的TALS质粒研究了5-溴尿嘧啶对体内核小体定位的影响。该质粒在与其α2操纵子结合的α2阻遏复合物的辅助下，能够稳定且精确地组装核小体。在操纵子附近的位点插入富含AT的序列会根据其长度和序列破坏依赖于核小体的定位。添加BrdU几乎完全通过特定的AT序列破坏了核小体定位。有效的富含AT的序列在聚丙烯酰胺凝胶电泳上迁移得更快，并且用5-溴尿嘧啶替代胸腺嘧啶会进一步加速它们的迁移率。由于这种特性表明DNA具有刚性构象，我们的结果表明5-溴尿嘧啶通过诱导A-型样DNA破坏核小体定位。
