Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4R2, Canada.
Nucleic Acids Res. 2019 Jan 25;47(2):716-728. doi: 10.1093/nar/gky1150.
Equal partitioning of the multi-copy yeast 2-micron plasmid requires association of plasmid proteins Rep1 and Rep2 with tandem repeats at the plasmid STB locus. To identify sequence elements required for these associations we generated synthetic versions of a 63-bp section of STB, encompassing one repeat. A single copy of this sequence was sufficient for Rep protein association in vivo, while two directly arrayed copies provided partitioning function to a plasmid lacking all other 2-micron sequences. Partitioning efficiency increased with increasing repeat number, reaching that conferred by the native STB repeat array. By altering sequences in synthetic repeats, we identified the TGCA component of a TGCATTTTT motif as critical for Rep protein recognition, with a second TGCA sequence in each repeat also contributing to association. Mutation of TGCATTTTT to TGTATTTT, as found in variant 2-micron STB repeats, also allowed Rep protein association, while mutation to TGCATTAAT impaired inheritance without abolishing Rep protein recognition, suggesting an alternate role for the T-tract. Our identification of sequence motifs required for Rep protein recognition provides the basis for understanding higher-order Rep protein arrangements at STB that enable the yeast 2-micron plasmid to be efficiently partitioned during host cell division.
多拷贝酵母 2 微米质粒的均等分配需要质粒蛋白 Rep1 和 Rep2 与质粒 STB 基因座上的串联重复序列结合。为了鉴定这些结合所需的序列元件，我们生成了 STB 的一个 63 个碱基对部分的合成版本，包含一个重复序列。该序列的单个拷贝足以在体内与 Rep 蛋白结合，而两个直接排列的拷贝则为缺乏所有其他 2 微米序列的质粒提供了分配功能。随着重复次数的增加，分配效率增加，达到了天然 STB 重复序列阵列所赋予的效率。通过改变合成重复序列中的序列，我们确定了 TGCATTTTT 基序中的 TGCA 成分对于 Rep 蛋白识别至关重要，每个重复中的第二个 TGCA 序列也有助于结合。在变体 2 微米 STB 重复序列中发现的 TGCATTTTT 突变为 TGTATTTT 也允许 Rep 蛋白结合，而突变为 TGCATTAAT 则在不废除 Rep 蛋白识别的情况下损害了遗传，这表明 T 链的替代作用。我们对 Rep 蛋白识别所需序列基序的鉴定为理解 STB 上更高阶的 Rep 蛋白排列提供了基础，这些排列使酵母 2 微米质粒能够在宿主细胞分裂过程中有效地分配。
