Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 2010 Apr 23;285(17):12612-9. doi: 10.1074/jbc.M109.082388. Epub 2010 Feb 23.
The replication terminator protein Fob1 of Saccharomyces cerevisiae is multifunctional, and it not only promotes polar replication fork arrest at the tandem Ter sites located in the intergenic spacer region of rDNA but also loads the NAD-dependent histone deacetylase Sir2 at Ter sites via a protein complex called RENT (regulator of nucleolar silencing and telophase exit). Sir2 is a component of the RENT complex, and its loading not only silences intrachromatid recombination in rDNA but also RNA polymerase II-catalyzed transcription. Here, we present three lines of evidence showing that the two aforementioned activities of Fob1 are independent of each other as well as functionally separable. First, a Fob1 ortholog of Saccharomyces bayanus expressed in a fob1Delta strain of S. cerevisiae restored polar fork arrest at Ter but not rDNA silencing. Second, a mutant form (I407T) of S. cerevisiae Fob1 retained normal fork arresting activity but was partially defective in rDNA silencing. We further show that the silencing defect of S. bayanus Fob1 and the Iota407Tau mutant of S. cerevisiae Fob1 were caused by the failure of the proteins to interact with two members of the S. cerevisiae RENT complex, namely S. cerevisiae Sir2 and S. cerevisiae Net1. Third, deletions of the intra-S phase checkpoint proteins Tof1 and Csm3 abolished fork arrest by Fob1 at Ter without causing loss of silencing. Taken together, the data support the conclusion that unlike some other functions of Fob1, rDNA silencing at Ter is independent of fork arrest.
酿酒酵母的复制终止蛋白 Fob1 具有多功能性，它不仅能促进串联 Ter 位点处的极性复制叉停滞，这些 Ter 位点位于 rDNA 的基因间间隔区，还能通过称为 RENT（核仁沉默和后期退出调节剂）的蛋白质复合物将 NAD 依赖性组蛋白去乙酰化酶 Sir2 加载到 Ter 位点。Sir2 是 RENT 复合物的一个组成部分，其加载不仅沉默 rDNA 内染色质重组，还抑制 RNA 聚合酶 II 催化的转录。在这里，我们提出了三条证据表明，Fob1 的上述两种功能彼此独立，并且在功能上是可分离的。首先，在酿酒酵母 fob1Δ 菌株中表达的酿酒酵母 bayanus 的 Fob1 同源物恢复了 Ter 处的极性叉停滞，但不能沉默 rDNA。其次，酿酒酵母 Fob1 的突变形式（I407T）保留了正常的叉停滞活性，但在 rDNA 沉默方面存在部分缺陷。我们进一步表明，酿酒酵母 bayanus Fob1 的沉默缺陷和酿酒酵母 Fob1 的 Iota407Tau 突变是由于这些蛋白无法与酿酒酵母 RENT 复合物的两个成员相互作用造成的，即酿酒酵母 Sir2 和酿酒酵母 Net1。第三，内 S 期检查点蛋白 Tof1 和 Csm3 的缺失消除了 Fob1 在 Ter 处的叉停滞，但不会导致沉默丧失。总之，这些数据支持以下结论：与 Fob1 的其他一些功能不同，Ter 处的 rDNA 沉默不依赖于叉停滞。
